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Sample GSM5726259

Query DataSets for GSM5726259

Status

Public on Feb 04, 2022

Title

mRNA-stim-well2

Sample type

SRA

Source name

Primary bulk T cells

Organism

Homo sapiens

Characteristics

stimulation: yes

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
features.tsv.gz
barcodes.tsv.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

E-mail(s)

zachary.steinhart@gladstone.ucsf.edu

Organization name

J. David Gladstone Institutes

Department

Gladstone-UCSF Institute of Genomic Immunology