title: rna1
organism: Homo sapiens
description: Protocols: Blood samples were obtained from four human healthy male adults, with a mean age of 26 (ages between 20-38). Peripheral blood mononuclear cells (PBMCs) were isolated by gradient density centrifugation, using Ficoll-Paque PLUS (Cytiva, 17144002). Naïve CD4+ T cells were isolated using EasySep Human Naïve CD4+ T Cell Isolation Kit (Stemcell technologies), according to manufacturer's indications.  Human naïve CD4+ T cells were seeded in tissue culturing 96 well plates, and stimulated with plate bound anti-human CD3 Antibody (clone OKT3, 5ng/mL, #317326, Biolegend) and aCD28 (clone CD28.2, 3 ng/mL, #302934, Biologened) at a density of 106 cells/mL. The cells were activated and differentiated in ImmunoCult-XF T Cell Expansion Medium, for 5 days. The list of cytokines and concentrations used for each subtype can be found in Supplementary Table S1. Activation and differentiation stimuli were added at the same time. On day 5, live cells were purified using flow cytometry. Cells were stained for viability discrimination using PI (P3566, Thermo Fisher Scientific), and were sorted for downstream purposes. Viability (92% on average) was then re-assessed by Trypan Blue staining (15250061, Thermo Fisher Scientific). Prior to mixing and single-cell library preparation, cells were treated to either (IL2, anti-CD28), (IL2,IL12,anti-IL4), (IL2,IL4,anti-ifng), (IL6,TGFb,IL23) or (IL2,TGFb,Atra) Dissociated cells were washed in ice-cold ATAC-seq resuspension buffer (RSB, 10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2), spun down, and resuspended in 100 mL ATAC-seq lysis buffer (RSB plus 0.1% NP-40 and 0.1% Tween-20 (Thermo Fisher). Lysis was allowed to proceed on ice for 5 min, then 900 mL RSB was added before spinning down again and resuspending in 50 mL 1X Nuclei Resuspension Buffer (10x Genomics). To assess nuclei purity and integrity after lysis, nuclei were stained with Trypan Blue (15250061, Thermo Fisher Scientific), and DAPI (D1306, Thermo Fisher Scientific), according to manufacturer recommendation. If necessary, cell concentrations were adjusted to equal ratios prior to starting single-cell GEM emulsion droplet generation with the ATAC-seq NextGEM kit (10x Genomics). Briefly, nuclei were incubated in a transposition mix. Transposed nuclei were then loaded into a Chromium Next GEM Chip J. 9,000 nuclei were loaded per lane, with a target recovery of 5,500 (expected doublet rate 4% - 4.8%). After GEM emulsion generation, reverse transcription, cDNA amplification and multiome sc library generation occurred, according to manufacturer specifications (10x genomic CG000338 Rev A). Part of the nucleic acid extraction protocol
attributes.tag: ENA first public
attributes.value: 2024-01-31
attributes.tag: INSDC center name
attributes.value: Laboratory of Molecular Infection Medicine Sweden (MIMS), Department of molecular biology, Umeå university
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-13711:rna1
attributes.tag: age
attributes.value: mixed
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: T cell
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: individual
attributes.value: mixed
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: blood
attributes.tag: replicate
attributes.value: 1
attributes.tag: sample name
attributes.value: E-MTAB-13711:rna1
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: male