ERS3961055 SAMEA6161044 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:07Z External Id SAMEA6161044 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:07Z INSDC status public Submitter Id E-MTAB-8498:23971_#1 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYA post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#1 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961056 SAMEA6161045 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:07Z External Id SAMEA6161045 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:07Z INSDC status public Submitter Id E-MTAB-8498:23971_#10 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#10 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961057 SAMEA6161046 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:07Z External Id SAMEA6161046 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:07Z INSDC status public Submitter Id E-MTAB-8498:23971_#100 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#100 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961058 SAMEA6161047 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:07Z External Id SAMEA6161047 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:07Z INSDC status public Submitter Id E-MTAB-8498:23971_#101 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#101 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961059 SAMEA6161048 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:07Z External Id SAMEA6161048 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:07Z INSDC status public Submitter Id E-MTAB-8498:23971_#102 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#102 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961060 SAMEA6161049 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161049 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#103 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#103 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961061 SAMEA6161050 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161050 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#104 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#104 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961062 SAMEA6161051 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161051 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#105 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#105 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961063 SAMEA6161052 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161052 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#106 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#106 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961064 SAMEA6161053 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161053 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#107 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#107 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961065 SAMEA6161054 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161054 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#108 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#108 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961066 SAMEA6161055 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161055 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#109 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#109 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961067 SAMEA6161056 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161056 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#11 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#11 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961068 SAMEA6161057 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161057 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#110 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#110 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961069 SAMEA6161058 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161058 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#111 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#111 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961070 SAMEA6161059 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161059 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#112 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#112 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961071 SAMEA6161060 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161060 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#113 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#113 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961072 SAMEA6161061 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161061 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#114 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#114 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961073 SAMEA6161062 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161062 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#115 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#115 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961074 SAMEA6161063 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161063 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#116 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#116 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961075 SAMEA6161064 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161064 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#117 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#117 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961076 SAMEA6161065 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161065 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#118 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#118 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961077 SAMEA6161066 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161066 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#119 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#119 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961078 SAMEA6161067 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161067 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#12 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#12 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961079 SAMEA6161068 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161068 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#120 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#120 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961080 SAMEA6161069 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161069 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#121 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#121 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961081 SAMEA6161070 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161070 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#122 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#122 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961082 SAMEA6161071 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161071 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#123 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#123 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961083 SAMEA6161072 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161072 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#124 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#124 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961084 SAMEA6161073 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161073 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#125 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#125 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961085 SAMEA6161074 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161074 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#126 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#126 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961086 SAMEA6161075 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161075 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#127 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#127 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961087 SAMEA6161076 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161076 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#128 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#128 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961088 SAMEA6161077 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161077 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#129 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#129 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961089 SAMEA6161078 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161078 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#13 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYB post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#13 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961090 SAMEA6161079 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161079 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#130 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#130 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961091 SAMEA6161080 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161080 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#131 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#131 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961092 SAMEA6161081 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161081 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#132 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#132 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961093 SAMEA6161082 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161082 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#133 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#133 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961094 SAMEA6161083 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161083 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#134 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#134 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961095 SAMEA6161084 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161084 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#135 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#135 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961096 SAMEA6161085 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161085 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#136 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#136 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961097 SAMEA6161086 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161086 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#137 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#137 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961098 SAMEA6161087 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161087 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#138 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#138 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961099 SAMEA6161088 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161088 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#139 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#139 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961100 SAMEA6161089 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161089 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#14 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYB post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#14 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961101 SAMEA6161090 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161090 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#140 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#140 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961102 SAMEA6161091 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161091 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#141 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#141 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961103 SAMEA6161092 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161092 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#142 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#142 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961104 SAMEA6161093 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161093 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#143 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#143 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961105 SAMEA6161094 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161094 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#144 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#144 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961106 SAMEA6161095 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161095 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#145 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#145 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961107 SAMEA6161096 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161096 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#146 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#146 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961108 SAMEA6161097 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161097 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#147 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#147 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961109 SAMEA6161098 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161098 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#148 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#148 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961110 SAMEA6161099 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161099 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#149 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#149 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961111 SAMEA6161100 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161100 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#15 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#15 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961112 SAMEA6161101 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161101 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#150 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#150 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961113 SAMEA6161102 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161102 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#151 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#151 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961114 SAMEA6161103 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:08Z External Id SAMEA6161103 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:08Z INSDC status public Submitter Id E-MTAB-8498:23971_#152 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#152 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961115 SAMEA6161104 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161104 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#153 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#153 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961116 SAMEA6161105 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161105 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#154 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#154 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961117 SAMEA6161106 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161106 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#155 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#155 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961118 SAMEA6161107 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161107 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#156 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#156 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961119 SAMEA6161108 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161108 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#157 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#157 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961120 SAMEA6161109 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161109 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#158 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#158 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961121 SAMEA6161110 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161110 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#159 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#159 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961122 SAMEA6161111 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161111 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#16 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#16 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961123 SAMEA6161112 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161112 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#160 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#160 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961124 SAMEA6161113 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161113 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#161 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#161 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961125 SAMEA6161114 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161114 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#162 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#162 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961126 SAMEA6161115 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161115 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#163 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#163 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961127 SAMEA6161116 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161116 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#164 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#164 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961128 SAMEA6161117 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161117 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#165 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#165 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961129 SAMEA6161118 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161118 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#166 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#166 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961130 SAMEA6161119 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161119 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#167 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#167 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961131 SAMEA6161120 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161120 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#168 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#168 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961132 SAMEA6161121 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161121 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#169 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#169 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961133 SAMEA6161122 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161122 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#17 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#17 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961134 SAMEA6161123 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161123 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#170 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#170 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961135 SAMEA6161124 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161124 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#171 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#171 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961136 SAMEA6161125 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161125 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#172 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#172 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961137 SAMEA6161126 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161126 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#173 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#173 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961138 SAMEA6161127 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161127 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#174 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#174 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961139 SAMEA6161128 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161128 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#175 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYA post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#175 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961140 SAMEA6161129 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161129 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#176 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#176 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961141 SAMEA6161130 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161130 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#177 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#177 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961142 SAMEA6161131 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161131 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#178 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYA post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#178 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961143 SAMEA6161132 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161132 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#179 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYA post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#179 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961144 SAMEA6161133 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161133 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#18 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#18 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961145 SAMEA6161134 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161134 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#180 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYA post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#180 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961146 SAMEA6161135 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161135 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#181 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYB post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#181 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961147 SAMEA6161136 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161136 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#182 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#182 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961148 SAMEA6161137 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161137 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#183 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#183 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961149 SAMEA6161138 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161138 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#184 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#184 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961150 SAMEA6161139 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161139 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#185 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#185 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961151 SAMEA6161140 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161140 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#186 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#186 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961152 SAMEA6161141 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161141 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#187 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#187 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961153 SAMEA6161142 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161142 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#188 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#188 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961154 SAMEA6161143 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161143 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#189 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYB post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#189 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961155 SAMEA6161144 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161144 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#19 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#19 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961156 SAMEA6161145 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161145 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#190 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYB post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#190 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961157 SAMEA6161146 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161146 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#191 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYB post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#191 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961158 SAMEA6161147 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161147 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#192 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYB post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#192 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961159 SAMEA6161148 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161148 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#193 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#193 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961160 SAMEA6161149 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161149 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#194 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#194 scientific_name Homo sapiens single cell well quality OK ERS3961161 SAMEA6161150 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161150 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#195 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#195 scientific_name Homo sapiens single cell well quality OK ERS3961162 SAMEA6161151 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161151 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#196 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#196 scientific_name Homo sapiens single cell well quality OK ERS3961163 SAMEA6161152 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161152 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#197 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#197 scientific_name Homo sapiens single cell well quality OK ERS3961164 SAMEA6161153 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161153 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#198 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#198 scientific_name Homo sapiens single cell well quality OK ERS3961165 SAMEA6161154 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161154 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#199 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#199 scientific_name Homo sapiens single cell well quality OK ERS3961166 SAMEA6161155 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161155 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#2 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#2 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961167 SAMEA6161156 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161156 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#20 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#20 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961168 SAMEA6161157 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161157 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#200 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#200 scientific_name Homo sapiens single cell well quality OK ERS3961169 SAMEA6161158 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161158 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#201 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#201 scientific_name Homo sapiens single cell well quality OK ERS3961170 SAMEA6161159 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161159 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#202 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#202 scientific_name Homo sapiens single cell well quality OK ERS3961171 SAMEA6161160 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161160 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#203 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#203 scientific_name Homo sapiens single cell well quality OK ERS3961172 SAMEA6161161 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161161 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#204 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#204 scientific_name Homo sapiens single cell well quality OK ERS3961173 SAMEA6161162 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161162 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#205 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#205 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961174 SAMEA6161163 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161163 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#206 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#206 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961175 SAMEA6161164 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161164 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#207 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#207 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961176 SAMEA6161165 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161165 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#208 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#208 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961177 SAMEA6161166 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161166 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#209 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#209 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961178 SAMEA6161167 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161167 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#21 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#21 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961179 SAMEA6161168 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161168 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#210 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#210 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961180 SAMEA6161169 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161169 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#211 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#211 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961181 SAMEA6161170 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:09Z External Id SAMEA6161170 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:09Z INSDC status public Submitter Id E-MTAB-8498:23971_#212 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#212 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961182 SAMEA6161171 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161171 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#213 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#213 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961183 SAMEA6161172 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161172 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#214 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#214 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961184 SAMEA6161173 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161173 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#215 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#215 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961185 SAMEA6161174 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161174 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#216 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#216 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961186 SAMEA6161175 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161175 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#217 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#217 scientific_name Homo sapiens single cell well quality OK ERS3961187 SAMEA6161176 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161176 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#218 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#218 scientific_name Homo sapiens single cell well quality OK ERS3961188 SAMEA6161177 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161177 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#219 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#219 scientific_name Homo sapiens single cell well quality OK ERS3961189 SAMEA6161178 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161178 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#22 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#22 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961190 SAMEA6161179 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161179 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#220 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#220 scientific_name Homo sapiens single cell well quality OK ERS3961191 SAMEA6161180 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161180 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#221 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#221 scientific_name Homo sapiens single cell well quality OK ERS3961192 SAMEA6161181 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161181 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#222 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#222 scientific_name Homo sapiens single cell well quality OK ERS3961193 SAMEA6161182 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161182 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#223 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#223 scientific_name Homo sapiens single cell well quality OK ERS3961194 SAMEA6161183 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161183 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#224 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#224 scientific_name Homo sapiens single cell well quality OK ERS3961195 SAMEA6161184 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161184 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#225 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#225 scientific_name Homo sapiens single cell well quality OK ERS3961196 SAMEA6161185 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161185 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#226 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#226 scientific_name Homo sapiens single cell well quality OK ERS3961197 SAMEA6161186 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161186 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#227 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#227 scientific_name Homo sapiens single cell well quality OK ERS3961198 SAMEA6161187 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161187 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#228 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#228 scientific_name Homo sapiens single cell well quality OK ERS3961199 SAMEA6161188 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161188 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#229 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#229 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961200 SAMEA6161189 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161189 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#23 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#23 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961201 SAMEA6161190 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161190 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#230 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#230 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961202 SAMEA6161191 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161191 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#231 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#231 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961203 SAMEA6161192 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161192 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#232 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#232 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961204 SAMEA6161193 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161193 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#233 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#233 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961205 SAMEA6161194 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161194 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#234 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#234 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961206 SAMEA6161195 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161195 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#235 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#235 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961207 SAMEA6161196 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161196 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#236 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#236 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961208 SAMEA6161197 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161197 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#237 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#237 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961209 SAMEA6161198 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161198 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#238 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#238 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961210 SAMEA6161199 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161199 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#239 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#239 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961211 SAMEA6161200 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161200 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#24 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#24 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961212 SAMEA6161201 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161201 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#240 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#240 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961213 SAMEA6161202 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161202 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#241 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#241 scientific_name Homo sapiens single cell well quality OK ERS3961214 SAMEA6161203 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161203 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#242 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#242 scientific_name Homo sapiens single cell well quality OK ERS3961215 SAMEA6161204 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161204 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#243 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#243 scientific_name Homo sapiens single cell well quality OK ERS3961216 SAMEA6161205 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161205 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#244 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#244 scientific_name Homo sapiens single cell well quality OK ERS3961217 SAMEA6161206 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161206 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#245 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#245 scientific_name Homo sapiens single cell well quality OK ERS3961218 SAMEA6161207 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161207 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#246 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#246 scientific_name Homo sapiens single cell well quality OK ERS3961219 SAMEA6161208 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161208 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#247 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#247 scientific_name Homo sapiens single cell well quality OK ERS3961220 SAMEA6161209 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161209 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#248 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#248 scientific_name Homo sapiens single cell well quality OK ERS3961221 SAMEA6161210 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161210 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#249 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#249 scientific_name Homo sapiens single cell well quality OK ERS3961222 SAMEA6161211 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161211 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#25 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#25 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961223 SAMEA6161212 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161212 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#250 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#250 scientific_name Homo sapiens single cell well quality OK ERS3961224 SAMEA6161213 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161213 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#251 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#251 scientific_name Homo sapiens single cell well quality OK ERS3961225 SAMEA6161214 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161214 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#252 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#252 scientific_name Homo sapiens single cell well quality OK ERS3961226 SAMEA6161215 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161215 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#253 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#253 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961227 SAMEA6161216 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161216 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#254 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#254 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961228 SAMEA6161217 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161217 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#255 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#255 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961229 SAMEA6161218 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161218 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#256 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#256 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961230 SAMEA6161219 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161219 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#257 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#257 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961231 SAMEA6161220 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161220 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#258 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#258 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961232 SAMEA6161221 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161221 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#259 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#259 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961233 SAMEA6161222 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161222 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#26 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#26 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961234 SAMEA6161223 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161223 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#260 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#260 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961235 SAMEA6161224 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161224 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#261 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#261 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961236 SAMEA6161225 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161225 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#262 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#262 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961237 SAMEA6161226 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161226 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#263 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#263 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961238 SAMEA6161227 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161227 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#264 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#264 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961239 SAMEA6161228 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161228 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#265 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#265 scientific_name Homo sapiens single cell well quality OK ERS3961240 SAMEA6161229 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161229 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#266 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#266 scientific_name Homo sapiens single cell well quality OK ERS3961241 SAMEA6161230 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161230 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#267 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#267 scientific_name Homo sapiens single cell well quality OK ERS3961242 SAMEA6161231 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161231 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#268 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#268 scientific_name Homo sapiens single cell well quality OK ERS3961243 SAMEA6161232 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161232 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#269 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#269 scientific_name Homo sapiens single cell well quality OK ERS3961244 SAMEA6161233 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161233 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#27 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#27 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961245 SAMEA6161234 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161234 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#270 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#270 scientific_name Homo sapiens single cell well quality OK ERS3961246 SAMEA6161235 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:10Z External Id SAMEA6161235 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:10Z INSDC status public Submitter Id E-MTAB-8498:23971_#271 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#271 scientific_name Homo sapiens single cell well quality OK ERS3961247 SAMEA6161236 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161236 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#272 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#272 scientific_name Homo sapiens single cell well quality OK ERS3961248 SAMEA6161237 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161237 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#273 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#273 scientific_name Homo sapiens single cell well quality OK ERS3961249 SAMEA6161238 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161238 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#274 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#274 scientific_name Homo sapiens single cell well quality OK ERS3961250 SAMEA6161239 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161239 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#275 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#275 scientific_name Homo sapiens single cell well quality OK ERS3961251 SAMEA6161240 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161240 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#276 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#276 scientific_name Homo sapiens single cell well quality OK ERS3961252 SAMEA6161241 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161241 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#277 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#277 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961253 SAMEA6161242 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161242 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#278 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#278 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961254 SAMEA6161243 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161243 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#279 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#279 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961255 SAMEA6161244 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161244 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#28 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#28 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961256 SAMEA6161245 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161245 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#280 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#280 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961257 SAMEA6161246 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161246 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#281 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#281 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961258 SAMEA6161247 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161247 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#282 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#282 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961259 SAMEA6161248 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161248 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#283 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#283 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961260 SAMEA6161249 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161249 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#284 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#284 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961261 SAMEA6161250 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161250 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#285 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#285 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961262 SAMEA6161251 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161251 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#286 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#286 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961263 SAMEA6161252 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161252 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#287 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#287 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961264 SAMEA6161253 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161253 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#288 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#288 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961265 SAMEA6161254 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161254 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#289 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#289 scientific_name Homo sapiens single cell well quality OK ERS3961266 SAMEA6161255 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161255 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#29 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#29 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961267 SAMEA6161256 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161256 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#290 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#290 scientific_name Homo sapiens single cell well quality OK ERS3961268 SAMEA6161257 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161257 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#291 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#291 scientific_name Homo sapiens single cell well quality OK ERS3961269 SAMEA6161258 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161258 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#292 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#292 scientific_name Homo sapiens single cell well quality OK ERS3961270 SAMEA6161259 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161259 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#293 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#293 scientific_name Homo sapiens single cell well quality OK ERS3961271 SAMEA6161260 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161260 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#294 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#294 scientific_name Homo sapiens single cell well quality OK ERS3961272 SAMEA6161261 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161261 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#295 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#295 scientific_name Homo sapiens single cell well quality OK ERS3961273 SAMEA6161262 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161262 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#296 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#296 scientific_name Homo sapiens single cell well quality OK ERS3961274 SAMEA6161263 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161263 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#297 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#297 scientific_name Homo sapiens single cell well quality OK ERS3961275 SAMEA6161264 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161264 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#298 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#298 scientific_name Homo sapiens single cell well quality OK ERS3961276 SAMEA6161265 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161265 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#299 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#299 scientific_name Homo sapiens single cell well quality OK ERS3961277 SAMEA6161266 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161266 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#3 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#3 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961278 SAMEA6161267 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161267 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#30 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#30 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961279 SAMEA6161268 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161268 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#300 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#300 scientific_name Homo sapiens single cell well quality OK ERS3961280 SAMEA6161269 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161269 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#301 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#301 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961281 SAMEA6161270 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161270 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#302 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#302 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961282 SAMEA6161271 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161271 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#303 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#303 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961283 SAMEA6161272 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161272 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#304 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#304 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961284 SAMEA6161273 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161273 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#305 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#305 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961285 SAMEA6161274 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161274 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#306 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#306 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961286 SAMEA6161275 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161275 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#307 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#307 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961287 SAMEA6161276 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161276 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#308 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#308 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961288 SAMEA6161277 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161277 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#309 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#309 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961289 SAMEA6161278 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161278 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#31 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#31 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961290 SAMEA6161279 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161279 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#310 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#310 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961291 SAMEA6161280 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161280 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#311 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#311 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961292 SAMEA6161281 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161281 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#312 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#312 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961293 SAMEA6161282 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161282 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#313 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#313 scientific_name Homo sapiens single cell well quality OK ERS3961294 SAMEA6161283 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161283 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#314 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#314 scientific_name Homo sapiens single cell well quality OK ERS3961295 SAMEA6161284 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161284 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#315 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#315 scientific_name Homo sapiens single cell well quality OK ERS3961296 SAMEA6161285 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161285 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#316 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#316 scientific_name Homo sapiens single cell well quality OK ERS3961297 SAMEA6161286 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161286 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#317 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type monocyte organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#317 scientific_name Homo sapiens single cell well quality OK ERS3961298 SAMEA6161287 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161287 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#318 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#318 scientific_name Homo sapiens single cell well quality OK ERS3961299 SAMEA6161288 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161288 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#319 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#319 scientific_name Homo sapiens single cell well quality OK ERS3961300 SAMEA6161289 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161289 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#32 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#32 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961301 SAMEA6161290 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161290 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#320 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#320 scientific_name Homo sapiens single cell well quality OK ERS3961302 SAMEA6161291 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161291 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#321 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#321 scientific_name Homo sapiens single cell well quality OK ERS3961303 SAMEA6161292 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161292 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#322 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#322 scientific_name Homo sapiens single cell well quality OK ERS3961304 SAMEA6161293 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161293 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#323 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#323 scientific_name Homo sapiens single cell well quality OK ERS3961305 SAMEA6161294 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161294 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#324 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#324 scientific_name Homo sapiens single cell well quality OK ERS3961306 SAMEA6161295 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161295 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#325 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#325 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961307 SAMEA6161296 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161296 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#326 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#326 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961308 SAMEA6161297 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161297 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#327 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#327 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961309 SAMEA6161298 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161298 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#328 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#328 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961310 SAMEA6161299 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161299 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#329 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#329 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961311 SAMEA6161300 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:11Z External Id SAMEA6161300 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:11Z INSDC status public Submitter Id E-MTAB-8498:23971_#33 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#33 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961312 SAMEA6161301 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161301 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#330 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#330 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961313 SAMEA6161302 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161302 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#331 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#331 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961314 SAMEA6161303 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161303 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#332 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#332 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961315 SAMEA6161304 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161304 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#333 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#333 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961316 SAMEA6161305 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161305 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#334 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#334 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961317 SAMEA6161306 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161306 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#335 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#335 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961318 SAMEA6161307 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161307 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#336 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#336 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961319 SAMEA6161308 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161308 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#337 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#337 scientific_name Homo sapiens single cell well quality OK ERS3961320 SAMEA6161309 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161309 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#338 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#338 scientific_name Homo sapiens single cell well quality OK ERS3961321 SAMEA6161310 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161310 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#339 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#339 scientific_name Homo sapiens single cell well quality OK ERS3961322 SAMEA6161311 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161311 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#34 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#34 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961323 SAMEA6161312 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161312 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#340 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#340 scientific_name Homo sapiens single cell well quality OK ERS3961324 SAMEA6161313 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161313 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#341 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#341 scientific_name Homo sapiens single cell well quality OK ERS3961325 SAMEA6161314 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161314 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#342 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#342 scientific_name Homo sapiens single cell well quality OK ERS3961326 SAMEA6161315 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161315 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#343 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#343 scientific_name Homo sapiens single cell well quality OK ERS3961327 SAMEA6161316 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161316 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#344 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#344 scientific_name Homo sapiens single cell well quality OK ERS3961328 SAMEA6161317 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161317 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#345 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#345 scientific_name Homo sapiens single cell well quality OK ERS3961329 SAMEA6161318 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161318 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#346 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#346 scientific_name Homo sapiens single cell well quality OK ERS3961330 SAMEA6161319 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161319 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#347 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#347 scientific_name Homo sapiens single cell well quality OK ERS3961331 SAMEA6161320 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161320 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#348 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#348 scientific_name Homo sapiens single cell well quality OK ERS3961332 SAMEA6161321 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161321 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#349 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#349 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961333 SAMEA6161322 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161322 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#35 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#35 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961334 SAMEA6161323 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161323 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#350 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#350 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961335 SAMEA6161324 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161324 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#351 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#351 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961336 SAMEA6161325 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161325 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#352 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#352 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961337 SAMEA6161326 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161326 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#353 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#353 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961338 SAMEA6161327 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161327 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#354 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#354 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961339 SAMEA6161328 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161328 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#355 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#355 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961340 SAMEA6161329 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161329 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#356 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#356 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961341 SAMEA6161330 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161330 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#357 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#357 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961342 SAMEA6161331 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161331 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#358 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#358 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961343 SAMEA6161332 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161332 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#359 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#359 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961344 SAMEA6161333 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161333 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#36 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#36 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961345 SAMEA6161334 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161334 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#360 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#360 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961346 SAMEA6161335 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161335 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#361 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#361 scientific_name Homo sapiens single cell well quality OK ERS3961347 SAMEA6161336 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161336 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#362 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#362 scientific_name Homo sapiens single cell well quality OK ERS3961348 SAMEA6161337 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161337 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#363 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#363 scientific_name Homo sapiens single cell well quality OK ERS3961349 SAMEA6161338 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161338 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#364 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#364 scientific_name Homo sapiens single cell well quality OK ERS3961350 SAMEA6161339 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161339 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#365 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#365 scientific_name Homo sapiens single cell well quality OK ERS3961351 SAMEA6161340 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161340 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#366 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#366 scientific_name Homo sapiens single cell well quality OK ERS3961352 SAMEA6161341 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161341 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#367 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#367 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961353 SAMEA6161342 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161342 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#368 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#368 scientific_name Homo sapiens single cell well quality OK ERS3961354 SAMEA6161343 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161343 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#369 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYC post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#369 scientific_name Homo sapiens single cell well quality OK ERS3961355 SAMEA6161344 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161344 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#37 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#37 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961356 SAMEA6161345 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161345 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#370 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#370 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961357 SAMEA6161346 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161346 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#371 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#371 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961358 SAMEA6161347 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161347 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#372 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYC post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#372 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961359 SAMEA6161348 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161348 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#373 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#373 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961360 SAMEA6161349 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161349 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#374 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#374 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961361 SAMEA6161350 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161350 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#375 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#375 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961362 SAMEA6161351 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161351 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#376 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#376 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961363 SAMEA6161352 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161352 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#377 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#377 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961364 SAMEA6161353 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161353 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#378 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#378 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961365 SAMEA6161354 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161354 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#379 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#379 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961366 SAMEA6161355 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161355 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#38 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#38 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961367 SAMEA6161356 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161356 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#380 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#380 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961368 SAMEA6161357 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161357 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#381 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#381 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961369 SAMEA6161358 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161358 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#382 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#382 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961370 SAMEA6161359 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161359 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#383 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#383 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961371 SAMEA6161360 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161360 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#384 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBY5 post analysis well quality fail run id 23971 sample name E-MTAB-8498:23971_#384 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961372 SAMEA6161361 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161361 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#39 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#39 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961373 SAMEA6161362 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161362 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#4 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#4 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961374 SAMEA6161363 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161363 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#40 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#40 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961375 SAMEA6161364 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161364 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#41 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#41 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961376 SAMEA6161365 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161365 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#42 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#42 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961377 SAMEA6161366 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161366 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#43 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#43 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961378 SAMEA6161367 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:12Z External Id SAMEA6161367 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:12Z INSDC status public Submitter Id E-MTAB-8498:23971_#44 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#44 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961379 SAMEA6161368 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161368 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#45 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#45 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961380 SAMEA6161369 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161369 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#46 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#46 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961381 SAMEA6161370 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161370 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#47 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#47 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961382 SAMEA6161371 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161371 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#48 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#48 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961383 SAMEA6161372 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161372 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#49 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#49 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961384 SAMEA6161373 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161373 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#5 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#5 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961385 SAMEA6161374 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161374 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#50 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#50 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961386 SAMEA6161375 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161375 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#51 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#51 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961387 SAMEA6161376 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161376 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#52 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#52 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961388 SAMEA6161377 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161377 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#53 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#53 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961389 SAMEA6161378 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161378 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#54 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#54 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961390 SAMEA6161379 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161379 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#55 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#55 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961391 SAMEA6161380 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161380 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#56 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#56 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961392 SAMEA6161381 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161381 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#57 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#57 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961393 SAMEA6161382 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161382 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#58 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#58 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961394 SAMEA6161383 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161383 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#59 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#59 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961395 SAMEA6161384 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161384 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#6 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#6 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961396 SAMEA6161385 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161385 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#60 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#60 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961397 SAMEA6161386 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161386 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#61 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#61 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961398 SAMEA6161387 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161387 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#62 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#62 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961399 SAMEA6161388 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161388 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#63 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#63 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961400 SAMEA6161389 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161389 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#64 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#64 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961401 SAMEA6161390 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161390 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#65 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#65 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961402 SAMEA6161391 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161391 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#66 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#66 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961403 SAMEA6161392 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161392 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#67 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#67 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961404 SAMEA6161393 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161393 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#68 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#68 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961405 SAMEA6161394 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161394 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#69 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#69 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961406 SAMEA6161395 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161395 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#7 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#7 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961407 SAMEA6161396 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161396 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#70 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#70 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961408 SAMEA6161397 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161397 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#71 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#71 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961409 SAMEA6161398 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161398 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#72 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#72 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961410 SAMEA6161399 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161399 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#73 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#73 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961411 SAMEA6161400 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161400 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#74 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#74 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961412 SAMEA6161401 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161401 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#75 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#75 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961413 SAMEA6161402 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161402 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#76 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#76 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961414 SAMEA6161403 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161403 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#77 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#77 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961415 SAMEA6161404 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161404 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#78 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#78 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961416 SAMEA6161405 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161405 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#79 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type Axl+Siglec-6+ dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#79 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961417 SAMEA6161406 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161406 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#8 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#8 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961418 SAMEA6161407 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161407 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#80 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#80 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961419 SAMEA6161408 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161408 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#81 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#81 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961420 SAMEA6161409 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161409 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#82 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#82 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961421 SAMEA6161410 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161410 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#83 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#83 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961422 SAMEA6161411 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161411 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#84 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#84 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961423 SAMEA6161412 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161412 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#85 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#85 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961424 SAMEA6161413 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161413 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#86 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#86 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961425 SAMEA6161414 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161414 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#87 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#87 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961426 SAMEA6161415 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161415 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#88 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#88 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961427 SAMEA6161416 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161416 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#89 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#89 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961428 SAMEA6161417 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161417 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#9 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#9 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961429 SAMEA6161418 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161418 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#90 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#90 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961430 SAMEA6161419 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161419 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#91 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#91 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961431 SAMEA6161420 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161420 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#92 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#92 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961432 SAMEA6161421 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161421 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#93 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#93 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961433 SAMEA6161422 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161422 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#94 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#94 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961434 SAMEA6161423 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161423 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#95 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#95 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961435 SAMEA6161424 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161424 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#96 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type monocyte organism part skin plate identity CF03YBYB post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#96 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961436 SAMEA6161425 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161425 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#97 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#97 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961437 SAMEA6161426 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161426 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#98 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#98 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961438 SAMEA6161427 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161427 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:23971_#99 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBYA post analysis well quality pass run id 23971 sample name E-MTAB-8498:23971_#99 scientific_name Homo sapiens single cell well quality OK stimulus house dust mite extract ERS3961439 SAMEA6161428 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161428 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:24013_#1 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#1 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961440 SAMEA6161429 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161429 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:24013_#10 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#10 scientific_name Homo sapiens single cell well quality OK ERS3961441 SAMEA6161430 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161430 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:24013_#100 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#100 scientific_name Homo sapiens single cell well quality OK ERS3961442 SAMEA6161431 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161431 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:24013_#101 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#101 scientific_name Homo sapiens single cell well quality OK ERS3961443 SAMEA6161432 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161432 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:24013_#102 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#102 scientific_name Homo sapiens single cell well quality OK ERS3961444 SAMEA6161433 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:13Z External Id SAMEA6161433 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:13Z INSDC status public Submitter Id E-MTAB-8498:24013_#103 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#103 scientific_name Homo sapiens single cell well quality OK ERS3961445 SAMEA6161434 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161434 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#104 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#104 scientific_name Homo sapiens single cell well quality OK ERS3961446 SAMEA6161435 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161435 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#105 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#105 scientific_name Homo sapiens single cell well quality OK ERS3961447 SAMEA6161436 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161436 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#106 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#106 scientific_name Homo sapiens single cell well quality OK ERS3961448 SAMEA6161437 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161437 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#107 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#107 scientific_name Homo sapiens single cell well quality OK ERS3961449 SAMEA6161438 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161438 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#108 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#108 scientific_name Homo sapiens single cell well quality OK ERS3961450 SAMEA6161439 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161439 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#109 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#109 scientific_name Homo sapiens single cell well quality OK ERS3961451 SAMEA6161440 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161440 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#11 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#11 scientific_name Homo sapiens single cell well quality OK ERS3961452 SAMEA6161441 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161441 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#110 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#110 scientific_name Homo sapiens single cell well quality OK ERS3961453 SAMEA6161442 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161442 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#111 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#111 scientific_name Homo sapiens single cell well quality OK ERS3961454 SAMEA6161443 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161443 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#112 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#112 scientific_name Homo sapiens single cell well quality OK ERS3961455 SAMEA6161444 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161444 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#113 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#113 scientific_name Homo sapiens single cell well quality OK ERS3961456 SAMEA6161445 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161445 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#114 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#114 scientific_name Homo sapiens single cell well quality OK ERS3961457 SAMEA6161446 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161446 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#115 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#115 scientific_name Homo sapiens single cell well quality OK ERS3961458 SAMEA6161447 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161447 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#116 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#116 scientific_name Homo sapiens single cell well quality OK ERS3961459 SAMEA6161448 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161448 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#117 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#117 scientific_name Homo sapiens single cell well quality OK ERS3961460 SAMEA6161449 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161449 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#118 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#118 scientific_name Homo sapiens single cell well quality OK ERS3961461 SAMEA6161450 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161450 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#119 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#119 scientific_name Homo sapiens single cell well quality OK ERS3961462 SAMEA6161451 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161451 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#12 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#12 scientific_name Homo sapiens single cell well quality OK ERS3961463 SAMEA6161452 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161452 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#120 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#120 scientific_name Homo sapiens single cell well quality OK ERS3961464 SAMEA6161453 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161453 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#121 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#121 scientific_name Homo sapiens single cell well quality OK ERS3961465 SAMEA6161454 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161454 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#122 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#122 scientific_name Homo sapiens single cell well quality OK ERS3961466 SAMEA6161455 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161455 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#123 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#123 scientific_name Homo sapiens single cell well quality OK ERS3961467 SAMEA6161456 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161456 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#124 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#124 scientific_name Homo sapiens single cell well quality OK ERS3961468 SAMEA6161457 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161457 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#125 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#125 scientific_name Homo sapiens single cell well quality OK ERS3961469 SAMEA6161458 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161458 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#126 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#126 scientific_name Homo sapiens single cell well quality OK ERS3961470 SAMEA6161459 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161459 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#127 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#127 scientific_name Homo sapiens single cell well quality OK ERS3961471 SAMEA6161460 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161460 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#128 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#128 scientific_name Homo sapiens single cell well quality OK ERS3961472 SAMEA6161461 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161461 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#129 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#129 scientific_name Homo sapiens single cell well quality OK ERS3961473 SAMEA6161462 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161462 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#13 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#13 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961474 SAMEA6161463 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161463 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#130 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#130 scientific_name Homo sapiens single cell well quality OK ERS3961475 SAMEA6161464 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161464 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#131 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#131 scientific_name Homo sapiens single cell well quality OK ERS3961476 SAMEA6161465 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161465 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#132 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#132 scientific_name Homo sapiens single cell well quality OK ERS3961477 SAMEA6161466 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161466 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#133 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#133 scientific_name Homo sapiens single cell well quality OK ERS3961478 SAMEA6161467 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161467 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#134 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#134 scientific_name Homo sapiens single cell well quality OK ERS3961479 SAMEA6161468 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161468 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#135 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#135 scientific_name Homo sapiens single cell well quality OK ERS3961480 SAMEA6161469 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161469 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#136 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#136 scientific_name Homo sapiens single cell well quality OK ERS3961481 SAMEA6161470 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161470 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#137 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#137 scientific_name Homo sapiens single cell well quality OK ERS3961482 SAMEA6161471 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161471 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#138 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type plasmacytoid dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#138 scientific_name Homo sapiens single cell well quality OK ERS3961483 SAMEA6161472 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161472 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#139 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#139 scientific_name Homo sapiens single cell well quality OK ERS3961484 SAMEA6161473 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161473 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#14 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#14 scientific_name Homo sapiens single cell well quality OK ERS3961485 SAMEA6161474 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161474 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#140 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#140 scientific_name Homo sapiens single cell well quality OK ERS3961486 SAMEA6161475 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161475 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#141 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type monocyte organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#141 scientific_name Homo sapiens single cell well quality OK ERS3961487 SAMEA6161476 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161476 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#142 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#142 scientific_name Homo sapiens single cell well quality OK ERS3961488 SAMEA6161477 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161477 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#143 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#143 scientific_name Homo sapiens single cell well quality OK ERS3961489 SAMEA6161478 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161478 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#144 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#144 scientific_name Homo sapiens single cell well quality OK ERS3961490 SAMEA6161479 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:20Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161479 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:20Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#145 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#145 scientific_name Homo sapiens single cell well quality OK ERS3961491 SAMEA6161480 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161480 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#146 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#146 scientific_name Homo sapiens single cell well quality OK ERS3961492 SAMEA6161481 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161481 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#147 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#147 scientific_name Homo sapiens single cell well quality OK ERS3961493 SAMEA6161482 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161482 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#148 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#148 scientific_name Homo sapiens single cell well quality OK ERS3961494 SAMEA6161483 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161483 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#149 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#149 scientific_name Homo sapiens single cell well quality OK ERS3961495 SAMEA6161484 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161484 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#15 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#15 scientific_name Homo sapiens single cell well quality OK ERS3961496 SAMEA6161485 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161485 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#150 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#150 scientific_name Homo sapiens single cell well quality OK ERS3961497 SAMEA6161486 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161486 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#151 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#151 scientific_name Homo sapiens single cell well quality OK ERS3961498 SAMEA6161487 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161487 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#152 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#152 scientific_name Homo sapiens single cell well quality OK ERS3961499 SAMEA6161488 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161488 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#153 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#153 scientific_name Homo sapiens single cell well quality OK ERS3961500 SAMEA6161489 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161489 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#154 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#154 scientific_name Homo sapiens single cell well quality OK ERS3961501 SAMEA6161490 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161490 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#155 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#155 scientific_name Homo sapiens single cell well quality OK ERS3961502 SAMEA6161491 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161491 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#156 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#156 scientific_name Homo sapiens single cell well quality OK ERS3961503 SAMEA6161492 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161492 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#157 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#157 scientific_name Homo sapiens single cell well quality OK ERS3961504 SAMEA6161493 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:14Z External Id SAMEA6161493 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:14Z INSDC status public Submitter Id E-MTAB-8498:24013_#158 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#158 scientific_name Homo sapiens single cell well quality OK ERS3961505 SAMEA6161494 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161494 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#159 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#159 scientific_name Homo sapiens single cell well quality OK ERS3961506 SAMEA6161495 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161495 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#16 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#16 scientific_name Homo sapiens single cell well quality OK ERS3961507 SAMEA6161496 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161496 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#160 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#160 scientific_name Homo sapiens single cell well quality OK ERS3961508 SAMEA6161497 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161497 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#161 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#161 scientific_name Homo sapiens single cell well quality OK ERS3961509 SAMEA6161498 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161498 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#162 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#162 scientific_name Homo sapiens single cell well quality OK ERS3961510 SAMEA6161499 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161499 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#163 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#163 scientific_name Homo sapiens single cell well quality OK ERS3961511 SAMEA6161500 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161500 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#164 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#164 scientific_name Homo sapiens single cell well quality OK ERS3961512 SAMEA6161501 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161501 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#165 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#165 scientific_name Homo sapiens single cell well quality OK ERS3961513 SAMEA6161502 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161502 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#166 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#166 scientific_name Homo sapiens single cell well quality OK ERS3961514 SAMEA6161503 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161503 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#167 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#167 scientific_name Homo sapiens single cell well quality OK ERS3961515 SAMEA6161504 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161504 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#168 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#168 scientific_name Homo sapiens single cell well quality OK ERS3961516 SAMEA6161505 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161505 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#169 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#169 scientific_name Homo sapiens single cell well quality OK ERS3961517 SAMEA6161506 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161506 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#17 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#17 scientific_name Homo sapiens single cell well quality OK ERS3961518 SAMEA6161507 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161507 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#170 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#170 scientific_name Homo sapiens single cell well quality OK ERS3961519 SAMEA6161508 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161508 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#171 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#171 scientific_name Homo sapiens single cell well quality OK ERS3961520 SAMEA6161509 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161509 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#172 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#172 scientific_name Homo sapiens single cell well quality OK ERS3961521 SAMEA6161510 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161510 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#173 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#173 scientific_name Homo sapiens single cell well quality OK ERS3961522 SAMEA6161511 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161511 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#174 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#174 scientific_name Homo sapiens single cell well quality OK ERS3961523 SAMEA6161512 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161512 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#175 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#175 scientific_name Homo sapiens single cell well quality OK ERS3961524 SAMEA6161513 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161513 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#176 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#176 scientific_name Homo sapiens single cell well quality OK ERS3961525 SAMEA6161514 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161514 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#177 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#177 scientific_name Homo sapiens single cell well quality OK ERS3961526 SAMEA6161515 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161515 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#178 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#178 scientific_name Homo sapiens single cell well quality OK ERS3961527 SAMEA6161516 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161516 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#179 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#179 scientific_name Homo sapiens single cell well quality OK ERS3961528 SAMEA6161517 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161517 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#18 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#18 scientific_name Homo sapiens single cell well quality OK ERS3961529 SAMEA6161518 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161518 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#180 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#180 scientific_name Homo sapiens single cell well quality OK ERS3961530 SAMEA6161519 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161519 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#181 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#181 scientific_name Homo sapiens single cell well quality OK ERS3961531 SAMEA6161520 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161520 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#182 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#182 scientific_name Homo sapiens single cell well quality OK ERS3961532 SAMEA6161521 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161521 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#183 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#183 scientific_name Homo sapiens single cell well quality OK ERS3961533 SAMEA6161522 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161522 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#184 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#184 scientific_name Homo sapiens single cell well quality OK ERS3961534 SAMEA6161523 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161523 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#185 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#185 scientific_name Homo sapiens single cell well quality OK ERS3961535 SAMEA6161524 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161524 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#186 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a- CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#186 scientific_name Homo sapiens single cell well quality OK ERS3961536 SAMEA6161525 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161525 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#187 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#187 scientific_name Homo sapiens single cell well quality OK ERS3961537 SAMEA6161526 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161526 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#188 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#188 scientific_name Homo sapiens single cell well quality OK ERS3961538 SAMEA6161527 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161527 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#189 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#189 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961539 SAMEA6161528 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161528 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#19 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type activated dendritic cell organism part skin plate identity CF03YBY3 post analysis well quality pass run id 24013 sample name E-MTAB-8498:24013_#19 scientific_name Homo sapiens single cell well quality OK ERS3961540 SAMEA6161529 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161529 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#190 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#190 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961541 SAMEA6161530 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161530 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#191 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#191 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961542 SAMEA6161531 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161531 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#192 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#192 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961543 SAMEA6161532 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161532 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#193 broker name ArrayExpress cell type control_well common name human developmental stage adult disease normal facs marker control_well inferred cell type not available organism part control_well plate identity CF03YBYL post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#193 scientific_name Homo sapiens single cell well quality not OK stimulus control_well ERS3961544 SAMEA6161533 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161533 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#194 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYL post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#194 scientific_name Homo sapiens single cell well quality OK ERS3961545 SAMEA6161534 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161534 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#195 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYL post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#195 scientific_name Homo sapiens single cell well quality OK ERS3961546 SAMEA6161535 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161535 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#196 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYL post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#196 scientific_name Homo sapiens single cell well quality OK ERS3961547 SAMEA6161536 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161536 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#197 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYL post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#197 scientific_name Homo sapiens single cell well quality OK ERS3961548 SAMEA6161537 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161537 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#198 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYL post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#198 scientific_name Homo sapiens single cell well quality OK ERS3961549 SAMEA6161538 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161538 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#199 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part skin plate identity CF03YBYL post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#199 scientific_name Homo sapiens single cell well quality OK ERS3961550 SAMEA6161539 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161539 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#2 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Hi BDCA2+ inferred cell type not available organism part blood plate identity CF03YBY9 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#2 scientific_name Homo sapiens single cell well quality OK ERS3961551 SAMEA6161540 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161540 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#20 broker name ArrayExpress cell type dendritic cell common name human developmental stage adult disease normal facs marker CD1a+ CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBY3 post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#20 scientific_name Homo sapiens single cell well quality OK ERS3961552 SAMEA6161541 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161541 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#200 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYL post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#200 scientific_name Homo sapiens single cell well quality OK ERS3961553 SAMEA6161542 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161542 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#201 broker name ArrayExpress cell type plasmacytoid dendritic cell common name human developmental stage adult disease normal facs marker CD123Int BDCA2+ inferred cell type not available organism part skin plate identity CF03YBYL post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#201 scientific_name Homo sapiens single cell well quality OK ERS3961554 SAMEA6161543 9606 Homo sapiens Protocols: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures. Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run. Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific). Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara). For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters. ENA-FIRST-PUBLIC 2019-12-01T04:05:21Z ENA-LAST-UPDATE 2019-11-07T08:27:15Z External Id SAMEA6161543 INSDC center name Wellcome Sanger Institute INSDC first public 2019-12-01T04:05:21Z INSDC last update 2019-11-07T08:27:15Z INSDC status public Submitter Id E-MTAB-8498:24013_#202 broker name ArrayExpress cell type conventional dendritic cell common name human developmental stage adult disease normal facs marker CD11c+ HLA-DR+ inferred cell type not available organism part skin plate identity CF03YBYL post analysis well quality fail run id 24013 sample name E-MTAB-8498:24013_#202 scientific_name Homo sapiens single cell well quality OK