RNA-Seq of human CD4 T cells
1e7 frozen CD4+ T cells were thawed and activated using human T cell activation beads in 10 mL of TCM. 24 hours later, the T cells were infected with a 10-15% v/v solution of 100x concentrated dCas9-ZIM3-mCherry lentivirus, facilitating the introduction of the CRISPR interference machinery. 24 hours after the CRISPRi infection, the cells were further infected with a 10-15% v/v solution of 100x concentrated gRNA virus designed to target specific variants of interest (Supplementary Table 30 for gRNA sequences). As a positive control we utilized a CD45 sgRNA to assess the efficacy of gene suppression. On day 2 post-guide infection, puromycin was added into the culture at a final concentration of 2.0 µg/mL in fresh TCM to select for transduced cells and adjust the cell density to 0.5 million cells/mL. On day 4, the puromycin concentration was reduced to 1.0 µg/mL to maintain selection. Cells were expanded for an additional 3-4 days. One day prior to cell sorting, flow cytometry analysis was performed to confirm the efficiency of CD45 knockdown (>85%) in mCherryhi / GFPhi cell populations. On day 7 or 8, a flow sorter was utilized to isolate at least 300k cells exhibiting high expression levels of mCherryhi / GFPhi. Post-sorting, cells were centrifuged to remove supernatant and immediately lysed using trizol reagent. Cell lysates were subsequently frozen at -80°C prior to RNA extraction. RNA extraction was carried out using Direct-zol RNA Microprep (ZYMO # R2063), following the manufacturer's instructions, to obtain RNA for bulk RNA-seq. Total RNA was added to reaction buffer from the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara, #634891), and reverse transcription was performed followed by PCR amplification to generate full length amplified cDNA. Sequencing libraries were constructed using the NexteraXT DNA library preparation kit with unique dual indexes (Illumina, FC-131-1096) to generate Illumina- 14 The copyright holder for this preprint this version posted October 12, 2024. ; https://doi.org/10.1101/2024.10.07.617092 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. compatible barcoded libraries. Libraries were pooled and quantified using a Qubit® Fluorometer (Life Technologies). Sequencing of pooled libraries was carried out on a NextSeq 2000 sequencer (Illumina) with paired-end 59-base reads, using a NextSeq P2 sequencing kit (Illumina) with a target depth of 5 million reads per sample.
NIAID-phs004072
lib98317