Comment[GEAAccession]	E-GEAD-740
MAGE-TAB Version	1.1
Investigation Title	5' CITE-seq for CD4+ cells and subpopulations
Experiment Description	To performe cellular indexing of transcriptomes and epitopes, we conducted CITE-seq. *** Related raw data are to be made available through the controlled-access database JGA (study accession JGAS000689, https://humandbs.dbcls.jp/hum0350-v1). ***
Experimental Design	cell type comparison design
Experimental Factor Name	isolate	cell_subtype	replicate
Experimental Factor Type	isolate	cell_subtype	replicate
Person Last Name	Komatsu	Oguchi	Murakawa
Person First Name	Shuichiro	Akiko	Yasuhiro
Person Affiliation	RIKEN-IFOM Joint Laboratory for Cancer Genomics  Center for Integrative Medical Sciences, RIKEN		
Person Roles	submitter	submitter	submitter
Public Release Date	2024-05-14
Protocol Name	P-GEAD-2544	P-GEAD-2545	P-GEAD-2546	P-GEAD-2547	P-GEAD-2548	P-GEAD-2549
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning and feature extraction protocol	normalization data transformation protocol
Protocol Description	Blood samples were collected into vacuum tubes (Venoject II, Terumo, Cat#VP-H100K). CD4+ T cells were isolated by the immunomagnetic negative selection method using the MACSxpress Whole Blood CD4 T Cell Isolation Kit, human (Miltenyi Biotec). As for T cell subpopulations, The cells were stained by the antibody cocktail. The cocktail contained 50 microlitter of Brilliant Stain Buffer (BD Biosciences) supplemented with fluorochrome- conjugated antibodies: 5 microlitter CD25 (clone BC96, AF488, BioLegend), 20 microlitter LAG-3 (polyclonal, PE, R&D), 5 microlitter CXCR5 (CD185) (clone RF8B2, PerCP-Cy5.5, BD Biosciences), 0.75 microlitter CD3 (clone UCHT1, PE-Cy7, BioLegend), 2.5 microlitter CD196 (CCR6) (clone 11A9, APC, BD Biosciences), 1 microlitter CD45RA (clone HI100, APC-Cy7, BioLegend), 5 microlitter CD183 (clone 1C6/CXCR3, BV421, BD Biosciences), 1 microlitter CD4 (clone RPA-T4, BV510, BD Biosciences), 5 microlitter CD194 (CCR4) (clone L291H4, BV605, BioLegend), 7.5 microlitter CD197 (CCR7) (clone G043H7, BV711, BioLegend), and 0.5 microlitter CD8 (clone RPA-T8, PE-CF594, BD Biosciences). The fluorescence data were acquired by using a FACSAria IIu Cell Sorter (BD Biosciences) and the sorted cells were collected.	Circulating bulk CD4 T cells and FACS-sorted heterogenous subpopulations were stained with TotalSeqTM-C Universal Cocktail, V1.0 (BioLegend). The cells were blocked with 2.5 microlitter of Human TruStain FcXTM blocking reagent (BioLegend) diluted in 22.5 microlitter of Cell Staining Buffer (BioLegend) for 10 min at 4 Celcius degree. The antibody cocktail (25 microlitter) was added, and cells were incubated for 30 min at 4 Celcius degree. Cells were washed 3 times with Cell Staining Buffer, filtered through a 40-microm FlowmiTM Cell Strainer (Bel-Art, H-B Instrument), and resuspended at 1.0 x 10^6 cells/ml in 1x PBS with 0.04% BSA (Invitrogen). Cells were loaded onto a Chromium Next GEM Chip K or Chip N (10x Genomics) using the V(D)J kit (v2) (10x Genomics).	Libraries were constructed according to the manufacturer's instructions (10x Genomics). Libraries were sequenced on an Illumina Novaseq 6000 sequencing platform using paired-end, dual-index sequencing with 150 cycles for read 1, 10 cycles for i7 index, 10 cycles for i5 index, and 150 cycles for read 2.	Libraries were constructed according to the manufacturer's instructions (10x Genomics). Libraries were sequenced on an Illumina Novaseq 6000 sequencing platform using paired-end, dual-index sequencing with 150 cycles for read 1, 10 cycles for i7 index, 10 cycles for i5 index, and 150 cycles for read 2.	Libraries were constructed according to the manufacturer's instructions (10x Genomics). Libraries were sequenced on an Illumina Novaseq 6000 sequencing platform using paired-end, dual-index sequencing with 150 cycles for read 1, 10 cycles for i7 index, 10 cycles for i5 index, and 150 cycles for read 2.	Reads were aligned to the Cell Ranger GRCh38 reference genome (refdata-gex-GRCh38-2020-A, 10x Genomics), and raw gene expression matrices and protein expression matrices were generated using the cellranger count function without the include introns option in Cell Ranger version 5.0.1.
SDRF File	E-GEAD-740.sdrf.txt
Comment[Number of channel]	single-channel
Comment[DBCLS]	The Data Access Committee of the Database Center for Life Science (DBCLS) approved that this personal data was made published according to the NBDC Guidelines for Human Data Sharing (https://humandbs.dbcls.jp/en/guidelines/data-sharing-guidelines) as the NBDC Research ID hum0350 and the application ID J-DS000641-001.
Comment[Array Design REF]	A-GEAD-11
Comment[AEExperimentType]	transcription profiling by array
Comment[BioProject]	PRJDB13816
Comment[Related study]	JGA:JGAS000689	NBDC:hum0350
Comment[Last Update Date]	2024-06-29