Comment[GEAAccession]	E-GEAD-743
MAGE-TAB Version	1.1
Investigation Title	3' single-nucleus RNA-seq as the multiome analysis of CD4+ cells and subpopulations
Experiment Description	We performed Chromium Single-Cell Multiome ATAC & Gene Expression on bulk CD4+ T cells to measure chromatin accessibility and 3' gene expression in parallel; then, we integrated the obtained data with 5' scRNA-seq data. To profile gene expression and enhancer activities at nucleotide resolution, we conducted 3'-end single-nucleus RNA-sequencing. *** 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-2562	P-GEAD-2563	P-GEAD-2564	P-GEAD-2565	P-GEAD-2566	P-GEAD-2567
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). For activated CD4+ T cells, these cells were kept in culture without any beads for 24 hr and then stimulated with Immunocult Human CD3/CD28 T cell activator Dynabeads for 48 hr. 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.	Cells collected on the same day were used partly for the 5' single cell (nuclei) assay and partly for the Multiome assay. Nuclei were isolated according to the manufacturer's instructions for the Low Cell Input Nuclei Isolation protocol (https://cdn.10xgenomics.com/image/upload/v1660261285/support-documents/CG000365_DemonstratedProtocol_NucleiIsolation_ATAC_GEX_Sequencing_RevC.pdf) with minor modifications. Nuclei were prepared either on ice or in a centrifuge at 4Celcius degree. Each centrifugation step was carried out for 5 min at 500 *g. Approximately 100,000 cells were washed in ice-cold 1% FBS in PBS, centrifuged, and then resuspended in 100 microlitter of lysis buffer (0.01% digitonin [Thermo Fisher Scientific, Cat#BN2006], 0.1% NP-40 [Sigma-Aldrich, Cat#NP40S-100ML], 0.1% Tween-20 [Bio-Rad, Cat#1662404], 10 mM Tris-HCl, pH 7.4 [Sigma-Aldrich, Cat#T2194], 10 mM NaCl [Nacalai Tesque, Cat#06900-14], 3 mM MgCl2 [Nippon Gene, Cat#310-90361], 1% BSA [Miltenyi Biotec, Cat#130-091-376], 1 mM DTT [Invitrogen, Cat#Y00147], and 1 U/microlitter RNase inhibitor [Sigma-Aldrich, Cat#3335402001] in nuclease-free water). The cells were incubated on ice for 1 min, and 400 microlitter of wash buffer (0.1% Tween-20, 10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 1% BSA, 1 mM DTT, and 1 U/microlitter RNase inhibitor in nuclease-free water) was added. The cells were centrifuged, the supernatant (495 microlitter) was removed, and 45 microlitter of 1* Nuclei Buffer supplemented with 1 mM DTT and 1 U/microlitter RNase inhibitor in nuclease-free water was added to the pellet without pipetting. The sample was centrifuged, and the nuclei were resuspended in 1* Nuclei Buffer, and were proceed to Multiome assays	Multiome assay was performed according to the manufacturer's instructions. Transposition was performed in a 10 microlitter reaction mixture at 37Celcius degree for 60 min, targeting up to 10,000 nuclei. Subsequently, nuclei were loaded onto a Chromium Chip J (10x Genomics) for GEM generation and barcoding. The post-GEM cleanup was followed by a pre-amplification step. Subsequently, each sample was divided into two fractions: one was allocated for the generation of a 3' snRNA-seq library, and the other for a snATAC-seq library. To enable multiplexing, both libraries were indexed using the Chromium i7 Sample Index N from the Set A kit (10x Genomics) for 3' snRNA-seq and the Chromium i7 Sample Index TT from the Set A kit (10x Genomics) for snATAC-seq.	The libraries were pooled and sequenced on an Illumina Novaseq 6000 platform using paired-end, dual-index sequencing with 150 cycles for read 1, 10 cycles for i7 index, 24 cycles for i5 index, and 150 cycles for read 2.	not applicable	Reads were aligned to the Cell Ranger GRCh38 reference genome (refdata-cellranger-arc-GRCh38-2020-A-2.0.0, 10x Genomics) and quantified using the cellranger-arc count function following the 10x Genomics guidelines.
SDRF File	E-GEAD-743.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