Multiplexed CROP-seq of Mac2a cells: sample2
Multiplexed single cell RNA sequencing was used to assess changes in gene expression after silencing of hit genes (selected from genome-wide CRISPR-Cas9 screens in Mac2a cells) and drug treatment. Mac2a cells harboring sgRNAs in CROPseq-Guide-Puro were pooled and plated in 24-well plate wells at 500 000 cells per well in 1 ml on cell culture medium. Cell pool 1 included idasanutlin hit gene sgRNAs (sgCDK6.1, sgCDK6.2, sgMDM4.2, sgPPM1D.1, sgPPM1D.2, sgCDKN1A.1, sgCDKN1A.2, sgTP53.1 and sgTP53.2) and two negative control sgRNAs (sgCtrl1 and sgCtrlCHR6). Cell pool 2 included Venetoclax, Navitoclax and Ruxolitinib hit gene sgRNAs (sgBAX.1, sgBAX.2, sgPMAIP.1 and sgPMAIP1.2) and the same two negative control sgRNAs. Cell pool 1 was treated with 300 nM idasanutlin and 0.1 % DMSO control. Cell pool 2 was treated with 100 nM navitoclax, 1000 nM venetoclax, 200 nM Ruxolitinib and 0.1% DMSO control. After 24 hours of incubation the cell numbers and viabilities were assessed. The drug or DMSO treated cells were stained with distinct TotalSeq-A0251 anti-human Hashtag antibodies. The cells were kept on ice and washed with cold PBS and resuspended in 100 l of Staining buffer, #420201, Biolegend). In each sample 10 l Fc Blocking reagent (FcX, BioLegend) was added and the cells were incubated for 10 minutes at 4C. An optimized amount (1 l / 0.5 g) of a distinct TotalSeq-A0251 anti-human Hashtag antibody (BioLegend) was added to each sample and the cells were incubated for 30 minutes at 4C. Cells were washed with Staining buffer and resuspeded in 0.5 ml of PBS + 0.04 % BSA. Cell numbers were calculated and equivalent number of cells were merged from each drug treatment: sequencing sample 1: cell line pool 1 treated with Idasanutlin and DMSO; sequencing sample 2: cell line pool 2 treated with Venetoclax, Navitoclax, Ruxolitinib and DMSO. The two sequencing samples were washed and resuspended in PBS + 0.04 % BSA at 2 x 106 cells/ml for sequencing. Single cell gene expression profiles were studied using 10x Genomics Chromium Single Cell 3 Gene Expression platform. The Chromium Single Cell 3RNAseq run and library preparation were done using the Chromium Next GEM Single Cell 3' Gene Expression version 3.1 Dual Index chemistry. The Sample libraries were sequenced on Illumina NovaSeq 6000 system using read lengths: 28bp (Read 1), 10bp (i7 Index), 10bp (i5 Index) and 90bp (Read 2). Data preprocessing was performed with 10x Genomics Cell Ranger v7.2.0 pipelines of which the cellranger mkfastq was used with Illumina bcl2fastq v2.2.0 to produce FASTQ (raw sequence data) files and cellranger count to perform alignment, filtering and UMI counting. Reads were aligned to the human reference genome (GRCh38) using the GENCODE v32/Ensembl98 transcriptome annotation. Count matrix for Hashtag oligonucleotides (HTO) was generated using the CITE-seq-Count-tool.