SRX5624853 GSM3702456 SRP190165 SRS4569982 GSM3702456 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702456 GSM3702456 GEO Accession GSM3702456 SRX5624854 GSM3702457 SRP190165 SRS4569984 GSM3702457 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702457 GSM3702457 GEO Accession GSM3702457 SRX5624855 GSM3702458 SRP190165 SRS4569983 GSM3702458 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702458 GSM3702458 GEO Accession GSM3702458 SRX5624856 GSM3702459 SRP190165 SRS4569985 GSM3702459 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702459 GSM3702459 GEO Accession GSM3702459 SRX5624857 GSM3702460 SRP190165 SRS4569986 GSM3702460 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702460 GSM3702460 GEO Accession GSM3702460 SRX5624858 GSM3702461 SRP190165 SRS4569987 GSM3702461 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702461 GSM3702461 GEO Accession GSM3702461 SRX5624859 GSM3702462 SRP190165 SRS4569989 GSM3702462 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702462 GSM3702462 GEO Accession GSM3702462 SRX5624860 GSM3702463 SRP190165 SRS4569988 GSM3702463 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702463 GSM3702463 GEO Accession GSM3702463 SRX5624861 GSM3702464 SRP190165 SRS4569990 GSM3702464 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702464 GSM3702464 GEO Accession GSM3702464 SRX5624862 GSM3702465 SRP190165 SRS4569992 GSM3702465 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702465 GSM3702465 GEO Accession GSM3702465 SRX5624863 GSM3702466 SRP190165 SRS4569991 GSM3702466 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702466 GSM3702466 GEO Accession GSM3702466 SRX5624864 GSM3702467 SRP190165 SRS4569993 GSM3702467 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702467 GSM3702467 GEO Accession GSM3702467 SRX5624865 GSM3702468 SRP190165 SRS4569994 GSM3702468 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702468 GSM3702468 GEO Accession GSM3702468 SRX5624866 GSM3702469 SRP190165 SRS4569995 GSM3702469 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702469 GSM3702469 GEO Accession GSM3702469 SRX5624867 GSM3702470 SRP190165 SRS4569997 GSM3702470 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702470 GSM3702470 GEO Accession GSM3702470 SRX5624868 GSM3702471 SRP190165 SRS4569996 GSM3702471 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702471 GSM3702471 GEO Accession GSM3702471 SRX5624869 GSM3702472 SRP190165 SRS4569998 GSM3702472 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702472 GSM3702472 GEO Accession GSM3702472 SRX5624870 GSM3702473 SRP190165 SRS4569999 GSM3702473 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702473 GSM3702473 GEO Accession GSM3702473 SRX5624871 GSM3702474 SRP190165 SRS4570000 GSM3702474 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702474 GSM3702474 GEO Accession GSM3702474 SRX5624872 GSM3702475 SRP190165 SRS4570001 GSM3702475 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702475 GSM3702475 GEO Accession GSM3702475 SRX5624873 GSM3702476 SRP190165 SRS4570002 GSM3702476 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702476 GSM3702476 GEO Accession GSM3702476 SRX5624874 GSM3702477 SRP190165 SRS4570003 GSM3702477 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702477 GSM3702477 GEO Accession GSM3702477 SRX5624875 GSM3702478 SRP190165 SRS4570005 GSM3702478 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702478 GSM3702478 GEO Accession GSM3702478 SRX5624876 GSM3702479 SRP190165 SRS4570004 GSM3702479 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702479 GSM3702479 GEO Accession GSM3702479 SRX5624877 GSM3702480 SRP190165 SRS4570006 GSM3702480 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702480 GSM3702480 GEO Accession GSM3702480 SRX5624878 GSM3702481 SRP190165 SRS4570007 GSM3702481 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702481 GSM3702481 GEO Accession GSM3702481 SRX5624879 GSM3702482 SRP190165 SRS4570008 GSM3702482 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702482 GSM3702482 GEO Accession GSM3702482 SRX5624880 GSM3702483 SRP190165 SRS4570009 GSM3702483 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702483 GSM3702483 GEO Accession GSM3702483 SRX5624881 GSM3702484 SRP190165 SRS4570010 GSM3702484 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702484 GSM3702484 GEO Accession GSM3702484 SRX5624882 GSM3702485 SRP190165 SRS4570012 GSM3702485 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702485 GSM3702485 GEO Accession GSM3702485 SRX5624883 GSM3702486 SRP190165 SRS4570011 GSM3702486 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702486 GSM3702486 GEO Accession GSM3702486 SRX5624884 GSM3702487 SRP190165 SRS4570013 GSM3702487 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702487 GSM3702487 GEO Accession GSM3702487 SRX5624885 GSM3702488 SRP190165 SRS4570014 GSM3702488 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702488 GSM3702488 GEO Accession GSM3702488 SRX5624886 GSM3702489 SRP190165 SRS4570015 GSM3702489 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702489 GSM3702489 GEO Accession GSM3702489 SRX5624887 GSM3702490 SRP190165 SRS4570016 GSM3702490 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702490 GSM3702490 GEO Accession GSM3702490 SRX5624888 GSM3702491 SRP190165 SRS4570017 GSM3702491 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702491 GSM3702491 GEO Accession GSM3702491 SRX5624889 GSM3702492 SRP190165 SRS4570019 GSM3702492 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702492 GSM3702492 GEO Accession GSM3702492 SRX5624890 GSM3702493 SRP190165 SRS4570018 GSM3702493 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702493 GSM3702493 GEO Accession GSM3702493 SRX5624891 GSM3702494 SRP190165 SRS4570020 GSM3702494 RNA-Seq TRANSCRIPTOMIC cDNA Mice were euthanized and popliteal and inguinal LNs were dissected. Cells were collected, filtered through a 70µm cell strainer, washed and maintained strictly at 4°C. Dendritic cells were enriched by negative selection. Cells were stained with FACS antibodies. 10^4-10^5 cells from each dendritic cell or T cell subpopulation were sorted in 40 ul of Lysis/Binding Buffer (Life technologies), lysed for 5 min and frozen at -80°C. Cell lysates were thawed and mRNA was captured with 12 ul of Dynabeads oligo(dT) (Life technologies), and washed according to manufacture guidelines. Purified messenger RNA was eluted at 70°C with 10 ul of 10 mM Tris-Cl pH 7.5 and stored at -80°C cDNA was generated from 1ul of mRNA of each sample. cDNA quantity in each sample was evaluated by qPCR for Actin B gene, and then equivalent amounts of mRNA of each sample were taken for RNAseq library construction. Library construction was performed in a 96-well plate format. First, to open secondary RNA structures and allow annealing of the RT primer, the samples were incubated at 72˚C for 3 min and immediately transferred to 4˚C. Then, RT reaction mix (10 mM DTT, 4 mM dNTP, 2.5 U/µl Superscript III RT enzyme in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) was added into each well of the 96-well plate and the reaction was mixed. The 96-well plate was then spun down and moved into a cycler (Eppendorf) for the following incubation: 2 min at 42˚C, 50 min at 50˚C, 5 min at 85˚C. Indexed samples with equivalent amount of cDNA were pooled.  The pooled cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20µl reaction, incubating for 2.5 h at 16˚C. The product was purified with 1.4x volumes of SPRI beads, eluted in 8 µl and in-vitro transcribed (with the beads) at 37˚C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37˚C and the amplified RNA (aRNA) purified with 1.2x volumes of SPRI beads. Library preparation for high-throughput sequencing: The aRNA was chemically fragmented into short molecules (median size ~200 nucleotides) by incubating 3 min at 70˚C in Zn2+ RNA fragmentation solution (Ambion) and purified with two volumes of SPRI beads. The aRNA (5 µl) was preincubated 3 min at 70˚C with 1 µl of 100 µM ligation adapter; then, 14 µl of a mix containing 9.5% DMSO, 1 mM ATP, 20% PEG8000 and 1 U/µl T4 ligase in 50 mM Tris HCl pH7.5, 10 mM MgCl2 and 1mM DTT was added. The reaction was incubated at 22˚C for 2 h. The ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent; reaction mix contains Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/µl RT enzyme) and a primer complementary to the ligated adapter. The reaction was incubated for 2 min at 42˚C, 45 min at 50˚C and 5 min at 85˚C.  The cDNA was purified with 1.5x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 µM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kapa Biosystems). The forward primer contains the Illumina P5-Read1 sequences and the reverse primer contains the P7-Read2 sequences. The amplified pooled library was purified with 0.7x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument (Agilent). MARS-Seq libraries were sequenced using an Illumina HiSeq 1500. 3' RNA-seq for digital gene expression quantitation NextSeq 500 gds 303702494 GSM3702494 GEO Accession GSM3702494