GSM7951427: Channel 15; Homo sapiens; OTHER
We chose the Jurkat E6 cell line expressing dCas9-KRAB as our model to study. We transduced them with a genome-wide CRISPRi CROP-seq library at a high multiplicity of infection (MOI). The FACS-measured percentage of GFP positive cells was 99.6%, translating to an estimated MOI of ~6. From experience, we know that real MOI is typically higher than what is estimated from GFP positivity rate. After infection, we split the cells into two populations, including untreated cells and activated cells (cells treated with anti-TCR and anti-CD28 antibodies for approximately 24 hours to stimulate TCR signaling). Cells were then labelled with cell hashing antibodies. Multiple labels were used for the activated population to help with cell multiplet detection. Cells were loaded on 16 channels of 10x Chromium X instrument. We loaded on 115,000 cells per channel and the expected recovery rate was 60,000 cells per channel, including 24% multiplets. Samples were pooled unequally before they were loaded on the Chromium X: 10% untreated cells, 90% treated cells. A sequencing library was prepared using 3'Chemistry with a targeted primer panel: custom multiplex PCR step to enrich for specific transcript. Libraries were sequenced on NovaSeq S4 PE100 in asymetric read mode (R1: 28 cycles; R2: 172 cycles), with PhiX concentration of 1%. The expected coverage is around 9,000 to 10,000 input reads per cell. The genome-wide CRISPRi sgRNA library was designed to target the transcription start site (TSS) coordinates, calculated from publicly available FANTOM CAGE peaks data. In total 18,595 genes were targeted, with 4 sgRNAs per gene. Additionally, we designed another CRISPRi library targeting 3,220 genes with 4 sgRNAs per gene. This library was designed using TSS data specific to Jurkat cells, calculated from publicly accessible Jurkat CAGE-seq datasets. Both libraries were combined into a final library targeting 3,220 genes with 8 sgRNAs per gene and 15,375 genes with 4 sgRNAs per gene.