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Sample GSM4678057

Query DataSets for GSM4678057

Status

Public on Oct 19, 2021

Title

Unstimulated Healthy Donor 2

Sample type

SRA

Source name

Unstimulated Healthy donor

Organism

Homo sapiens

Characteristics

bach2 genotype: Wildtype (Healthy Donor)
cell type: Isolated and Treated Memory CD4+ Cells (Negative Magnetic Selection)
treatment: None
sequencing method: SE 50bp RNA-seq, HiSeq

Treatment protocol

Memory CD4 T Cell Isolation, Culture, and Treatment: Human PBMCs were purified from either anonymized leukodepletion cones (Blood Transfusion Service, NHS Blood and Transplantation) or healthy volunteer whole blood packs/buffy coats from the NIH Blood Bank and from fresh blood of patients. Leukodepletion cones were diluted 1 in 4 with sterile PBS and layered onto 15mL of lymphoprep (Axis-Shield). Whole blood packs/buffy coats were diluted 1 in 2 with sterile PBS onto 15mL Lymphocyte Separation Medium (25-072-CV LSM, Corning). Cells were separated by centrifugation at 20C for 20 min, with slow acceleration and no brakes and PBMC collected by harvesting interface cells. Human memory CD4+ T cells were used for RNA-seq, CUT&RUN, and CUT&Tag experiments as these cells express the VDR, while naïve T cells require pre-activation first. Human memory CD4+ T cells were isolated either from PBMCs using Miltenyi Memory CD4+ T cell Isolation Kit Human (130091893) or StemCell EasySep Human Memory CD4+ T Cell Enrichment Kit (19157) according to the manufacturer. Cells were cultured in Lonza X-VIVO-15 Serum-free Hematopoietic Cell Medium (04-418Q) supplemented with 50 IU/mL penicillin, 50µg/mL streptomycin (Invitrogen), 2mM L-glutamine (PAA Labarotories) at 37C 5% CO2. CD4+ memory T cells were cultured in a 96 well U bottom plate (Greiner) at a cell density of one million cells/mL in a total volume of 200µL. CD4+ cells were activated using Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (11131D) at a cell to bead ratio of 4:1. Cells were additionally cultured in the presence or absence of 1α,25-Dihydroxyvitamin D3 (Enzo Life Sciences), reconstituted in 99.8% ethanol (Sigma-Aldrich), used at 10nM unless indicated. 99.8% ethanol was used as a carrier control at the same concentration.

Extracted molecule

polyA RNA

Extraction protocol

Isolation of mRNA and RNA-seq: Memory CD4+ T cells, including from BACH2 WT and L24P patient samples, were isolated and activated with or without VitD treatment as described. 600,000 unactivated cells, or cells activated for 48 or 72 hrs, were pelleted at 300g for 5 min. RNA was stabilized by lysing cells in 100µL of RNAqueous lysis buffer, snap-freezing on dry-ice, followed by storage at -80C and isolation of total RNA by the RNAqueous-Micro kit (AM1931, Thermo Fisher Scientific) as per manufacturers guidelines. 1µg total RNA for each sample was subjected to NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490). Resulting mRNA was prepared for RNA-seq using a NEB Ultra II RNA Sequencing Library Prep Kit (New England Biolabs) as per the manufacturer's guidelines.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Description

processed data file: Mem_CD4.HealthyDonor_VitD_RNAseq.RPKMs.txt
NoStim.HD2.RNAseq

Data processing

RNA-seq Data Processing and Analysis: The expression level of all genes in all RNA-seq libraries was quantified by ‘rsem-calculate-expression’ in RSEM v1.3.1 with parameters ‘--bowtie-n 1 --bowtie-m 100 --seed-length 28 --bowtie-chunkmbs 1000’. The bowtie index for RSEM alignment was generated by ‘rsem-prepare-reference’ on all RefSeq genes, downloaded from UCSC table browser in April 2017. EdgeR/v3.26.8 was used to normalize gene expression among all libraries and identify differentially expressed genes among samples. Analysis of microarray data sourced from GSE119416 was carried out using Partek Genomics Suite (Partek, Inc.). Differentially expressed gene thresholds were defined using the following criteria: at least 1.5-fold change in either direction at p-value<0.05 for microarray analysis; at least 1.75-fold change in either direction at FDR<0.05 for RNA-seq analysis. Genome_build: GRCh37; hg19. Supplementary_files_format_and_content: RNA-seq data presented as RPKMs in tablature format.

Submission date

Jul 20, 2020

Last update date

Oct 19, 2021

Contact name

Behdad (Ben) Afzali Khoshkbijari

E-mail(s)

ben.afzali@nih.gov

Phone

301-443-2055

Organization name

National Institutes of Diabetes, Digestive, and Kidney Diseases