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Sample GSM5726254 Query DataSets for GSM5726254
Status Public on Feb 04, 2022
Title mRNA-nostim-well1
Sample type SRA
Source name Primary bulk T cells
Organism Homo sapiens
Characteristics stimulation: none
Treatment protocol One day after activation, T cells were infected with dCas9-VP64 (CRISPRa) lentivirus. Two days after activation, T cells were split into two populations and infected with a 154 sgRNA Perturb-seq library an MOI of 0.3. 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 harvested, spun down and resuspended at 2e6 cells/ml X-VIVO 15 without supplements. Next day, cells were split into two conditions: 1) re-stimulated with anti-CD3/CD28/CD2 immunocult or 2) left. 24 hours later cells were sorted for mCherry+ (marking dCas9-VP64) before droplet generation.
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 polyA RNA
Extraction protocol After sorting, cells were processed with the 10x Genomics Chromium Next GEM Single Cell 3′ Reagent Kit v3.1 with Feature Barcoding technology for CRISPR Screening, following manufacturer’s protocol (CG000205 Rev D). Before loading the Chromium chip, sorted cells from two blood donors were normalized to 1000 cells/µl and mixed at a 1:1 ratio, for each condition. Twenty µl of cell suspension was loaded into four replicate wells per condition, for a total 80,000 cells loaded per condition.
Libraries were constructed using the 10x Genomics Chromium Next GEM Single Cell 3′ Reagent Kit v3.1 with Feature Barcoding technology for CRISPR Screening, following manufacturer’s protocol (CG000205 Rev D). Final sgRNA sequencing libraries were further purified for the correct size fragment by 4% agarose E-Gel EX Gels (ThermoFisher Scientific) and gel extracted using the NucleoSpin Gel and PCR Clean-up Mini kit (Machery-Nagel).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Description matrix.mtx.gz
Data processing Sequencing reads were demultiplexed with bcl2fastq. Alignments and count aggregation of gene expression and sgRNA reads were completed with Cell Ranger version 6.1.1. Gene expression and sgRNA reads were aligned using cellranger count, with default settings. Gene expression reads were aligned to the “refdata-gex-GRCh38-2020-A” human transcriptome reference downloaded from 10x Genomics. sgRNA reads were aligned to the Perturb-seq library using the pattern (BC)GTTTAAGAGCTATG. Counts were aggregated with cellranger aggr with default arguments. To assign sgRNAs to cells, cellranger count output files “protospacer_calls_per_cell.csv” were manually aggregated in R using the bind_rows() function from the dplyr package to generate the “cellranger-guidecalls-aggregated-unfiltered.txt” file.
Genome_build: GRCh38-2020-A
Supplementary_files_format_and_content: cellranger aggr filtered feature barcode matrix output files (matrix.mtx.gz, features.tsv.gz, barcodes.tsv.gz), which can be loaded into Seurat or other similar software for scRNA-seq analysis
Supplementary_files_format_and_content: cellranger count “protospacer_calls_per_cell.csv" files manually aggregated across wells (cellranger-guidecalls-aggregated-unfiltered.txt)
Submission date Dec 09, 2021
Last update date Feb 04, 2022
Contact name Zachary Steinhart
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 GPL24676
Series (1)
GSE190604 CRISPR activation and interference screens decode stimulation responses in primary human T cells [CRISPRa Perturb-seq]
BioSample SAMN23829590
SRA SRX13372033

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

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