|
Status |
Public on May 13, 2021 |
Title |
10_Donor3_sgControl_Stim |
Sample type |
SRA |
|
|
Source name |
Primary CD4+ T cells
|
Organism |
Homo sapiens |
Characteristics |
cell type: Primary CD4+ T cells sgrna: No-Target Control treatment: anti-CD3/CD28/CD2 Immunocult
|
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.
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|
|
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 |
SAMN19110399 |
SRA |
SRX10863209 |