title: GSM8549456: CD4_Perturb-CITE-seq_CRISPR_04; Homo sapiens; OTHER
library_construction_protocol: On the 10th day after initial isolation, the samples were collected for 10X single cell sequencing. First, cells from each donor within the same stimulation and cell type condition were pooled at equal concentrations. Sorting was performed to isolate live GFP+ cells from each condition. Sorted cells were processed according to the Chromium Next GEM Single Cell 5' HT Reagent Kits v2 (Dual Index) with Feature Barcode technology for CRISPR Screening and Cell Surface Protein guide User Guide, CG000513. In brief, sorted cells were pelleted and washed once with Cell Staining Buffer (Biolegend, #420201). Next, the samples were blocked with Human TruStain FcX™ Fc Blocking reagent (Biolegend, #422302). Meanwhile, TotalSeq™-C Human Universal Cocktail V1.0 (Biolegend, #399905) was prepared using Cell Staining Buffer (Biolegend, #420201) and TotalSeq™-C0251 anti-human Hashtag Antibodies 1-4 (Biolegend, #394661) were added to aliquots of the cocktail. HTOs were added to label the following populations: Treg Resting-1, Treg Stimulated- 2, Teff Resting-3, Teff Stimulated- 4. After blocking, cells were stained with TotalSeq-C cocktail including one Hashtag per cell and stimulation condition. After staining, the cells were washed three times in Cell Staining Buffer. The samples were then resuspended in PBS with 1% BSA (Gibco) for final counting. The resulting samples were pooled across conditions and approximately 65,000 cells per well were loaded into 8 wells of a Chromium Next GEM Chip N Single Cell Kit (10X Genomics, #1000375) for GEM generation. The samples were prepared for sequencing using the Chromium Next GEM Single Cell 5' HT Kit v2 (#1000374), 5' Feature Barcode Kit, (#1000256), and 5' CRISPR Kit (#1000451) according to the manufacturer's protocol. GEM generation and library preparation was performed by the Gladstone Genomics Core. The resulting libraries were sequenced using a NovaSeqX Series 10B flowcell (Illumina, #20085595) at the UCSF CAT.