Single cell RNA-seq of human CD4 T cells
5 million CD4+ T cells were thawed and cultured in 5 mL of TCM supplemented with human T cell activation beads for 48 hours. T cells were then infected with a 2-4% (v/v) solution of 100x concentrated dCas9-ZIM3-mCherry lentivirus to introduce the CRISPR interference machinery. 24 hours later, the cells were washed twice with PBS and subsequently infected with an 8% (v/v) solution of 100x concentrated CRISPR-QTL virus, which targets specific variants of interest. 5% of gRNAs contained within the library targeted CD45 as a positive control to evaluate gene suppression efficacy. By day 2 post-library infection, puromycin was added to the culture at a final concentration of 2.0 µg/mL in fresh TCM to select for library transduced cells, and the cell density was adjusted to 0.5 million cells/mL. At 4 days post-infection (4dpi), the cells were resuspended in 20 mL of TCM containing 1.0 µg/mL of puromycin to maintain selection pressure. On days 10 to 12 post-infection, the cells were harvested and divided into six groups. They were stained with anti-CD3, anti-CD4, and anti-CD45 antibodies, as well as a live/dead dye. Six hashtag antibodies were also used to separate donors and to enable superloading of the 10X controller. Prior to sorting, six groups of cells were pooled, and then sorted. 150K (v1 – 1 donor) and 330K (v2 – 2 donors) mCherryhi/GFPhi cells (CD3+/CD4+/live/CD45+ pre-gated) were sorted using a FACS cell sorter. The sorted cells were promptly loaded onto two (v1) or six (v2) channels on the 10x Chromium X controller (10x Genomics) according to the manufacturer's protocol, with a target capture of 20,000 (library 1) and 12,500 (library 2) cells per channel. Sequencing libraries were generated using the Chromium Next GEM Single Cell 5′ Kit v2 (10x Genomics, #1000265). Gene expression, CRISPR, and feature barcoding libraries were pooled at a 4:1:1 ratio and treated with Illumina Free Adapter Blocking Reagent (Illumina, #20024144). Sequencing of pooled libraries was carried out on a NextSeq 2000 sequencer (Illumina), using a NextSeq P3 flowcell (Illumina) for v1 or sequenced on a Nova-seq X Plus 25B flow cell. Basecalls were processed to FASTQs on BaseSpace (Illumina).
NIAID-phs004072
crispr3