title: Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens
design: Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens
library_name: Tcell_CRISPR_35_p
library_construction_protocol: The human biological samples in this study were sourced ethically and their research use was in accord with the terms of the informed consent under an IRB/EC approved protocol. Briefly, mononuclear cells from circulating blood were removed by apheresis. From these leukopaks, primary human CD4+ T cells were enriched using CliniMACS Prodigy setup and human CD4 MicroBeads (Miltenyi, 130-045-101). Following positive selection, cells were aliquoted and cryopreserved. The screen dataset contains two different scRNAseq runs: the first one (8 channels of a 10X Genomics chip B) was performed with fresh cells at the end of the CRISPRi experiment, 10 days post gRNA library transduction; the second one (32 channels across four 10XGenomics chips B) was performed using cells from the same experiment that were cryopreserved 10 days after gRNA transduction and thawed in puromycin-containing media (2 ug/ml) three days before the 10X Genomics run. To remove dead cells and debris, the cell suspension was treated with Lymphoprep (Axis-Shield, 12HHS09) the day before loading the cells into the 10X Chromium Controller. Primary CD4+ T cell vials were thawed at the time of the experiments and cells cultured in RPMI 1640 (Gibco, 11340892) supplemented with 10% heat-inactivated FBS, 1x Glutamax (Thermo Fisher Scientific, 35050061), 1mM Sodium Pyruvate (Thermo Fisher Scientific, 11360088), 5mM HEPES (Thermo Fisher Scientific, 15630080), 1x non-essential aminoacids (Thermo Fisher Scientific, 11140035), 1x Penicillin/streptomycin (Thermo Fisher Scientific, 15070063), 55 µM 2-mercaptoethanol (Thermo Fisher Scientific, 21985023), and 15 ng/mL of recombinant human interleukin 2 (IL-2) (Peprotech, 200-02). Cells were kept in a humidified 5% CO2 atmosphere at 37°C. Primary human CD4+ T cells expressing CBh-ZIM3-dCas9-Bsd were activated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific, 11131D) at 1:1 cell:bead ratio and, 16-20h later, two million cells were transduced with the pooled CRISPRi CROPseq gRNA library at a <0.3 MOI. The transduction was performed in growth media supplemented with 5mM of HEPES (Thermo Fisher Scientific, 15630080) and spinoculation was carried out at 800 x g for 1h at 37˚C. 48h later, cells were selected with 2 ug/mL puromycin for 4 days, and selection was verified by ZsGreen expression in >95% of the cells by flow cytometry (Cytoflex S, Beckman Coulter). Cells were cultured in growth media for an additional 6 days before processing them for scRNA-seq. 10x Genomics Chromium Single Cell 3' Reagent Kit (v3 chemistry) 3' gene expression libraries (v3) were prepared following manufacturer's instructions (10X Genomics, PN-1000075). gRNA amplicon libraries were generated from amplified cDNA following the protocol described in (Alda-Catalinas et al. 2021).