NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM5291011 Query DataSets for GSM5291011
Status Public on May 13, 2021
Title 05_Donor2_sgControl_NoStim
Sample type SRA
 
Source name Primary CD4+ T cells
Organism Homo sapiens
Characteristics cell type: Primary CD4+ T cells
sgrna: No-Target Control
treatment: None
Treatment protocol One day after activation, Bulk T cells were transduced with mCherry-2A-dCas9-VP64 lentivirus. Two days after activation, T cells were split into two populations and transduced with either a non-targeting control, or FOXQ1 transcriptional start site targeting sgRNA lentivirus. The following day 2ug/ml puromycin was added to the culture medium and cells were passaged every two days maintaining a minimum concentration of 0.3e6cells/ml. Eight days after initial activation, cells were sorted into CD4+mCherry+ populations and resumed culture in X-VIVO 15 without supplements. The next day cells were treated with 6.25ul/ml anti-CD3/CD28/CD2 Immunocult (Stim) or left untreated (NoStim). Cells were harvest 24 hours after stimulation.
Growth protocol Primary human bulk T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641). Primary T cell activation was done by anti-humanCD3/CD28 CTS dynabeads (Fisher Scientific cat 40203D) at a 1:1 cell to bead ratio at 1e6 cells/ml.
Extracted molecule total RNA
Extraction protocol Cells were lysed and RNA was purified using Quick-RNA Microprep kit (Zymo Research) without the optional in-well DNase treatment step. Purified RNA was treated with TURBO DNase (Thermofisher Scientific) to remove potential contaminating DNA. RNA was subsequently purified using RNA Clean & Concentrator-5 kit (Zymo Research). RNA quality control was performed using an RNA ScreenTape assay (Agilent), with all samples having a RNA integrity number > 7.
RNA-seq libraries were prepared using the Illumina Stranded mRNA Prep kit, with 100ng of input RNA, following manufacturer's recommended protocol.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Data processing Adapters were trimmed from fastq files using cutadapt version 2.10 with default settings keeping a minimum read length of 20 bp.
Reads were mapped to the human genome GRCh38 keeping only uniquely mapping reads using STAR version 2.7.5b with the following settings “--outFilterMultimapNmax 1”.
Reads overlapping genes were then counted using featureCounts version 2.0.1 with the following settings “-s 2” and using the Gencode version 35 basic transcriptome annotation.
Genome_build: GRCh38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
 
Submission date May 11, 2021
Last update date May 14, 2021
Contact name Zachary Steinhart
E-mail(s) zachary.steinhart@gladstone.ucsf.edu
Organization name J. David Gladstone Institutes
Department Gladstone-UCSF Institute of Genomic Immunology
Lab Marson Lab
Street address 1650 Owens St
City San Francisco
State/province CA
ZIP/Postal code 94158
Country USA
 
Platform ID GPL18573
Series (2)
GSE174284 CRISPR activation and interference screens decode stimulation responses in primary human T cells [Bulk RNA-seq]
GSE174292 CRISPR activation and interference screens decode stimulation responses in primary human T cells
Relations
BioSample SAMN19110409
SRA SRX10863204

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap