CROP-seq raw feature barcode matrices

Multiplexed single cell RNA sequencing was used to assess changes in gene expression after p53 pathway hit gene silencing and drug treatment. Mac2a cells harboring sgRNAs in CROPseq-Guide-Puro cell pool 1 (CROPSeq1) 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) and was treated with 300 nM idasanutlin and 0.1 % DMSO control for 24h. Cell pool 2 (CROPSeq2) included Venetoclax, Navitoclax and Ruxolitinib hit gene sgRNAs (sgBAX.1, sgBAX.2, sgPMAIP.1 and sgPMAIP1.2) and the same two negative control sgRNAs and was treated with 100 nM navitoclax, 1000 nM venetoclax, 200 nM Ruxolitinib and 0.1 % DMSO control for 24h. The drug or DMSO treated cells were stained with distinct TotalSeq™-A0251 anti-human Hashtag antibodies after which equivalent number of cells were merged from each drug treatment: cell line pool 1 treated with Idasanutlin and DMSO; cell line pool 2 treated with Venetoclax, Navitoclax, Ruxolitinib and DMSO. 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. CROP-seq results were analyzed using command-line tools (GNU bash 4.4.20, Awk 4.2.1), and R version 4.3.3 with Bioconductor 3.22. Documentation of the workflow, including code and intermediate results, is available at https://github.com/HRUH-Mustjoki-Lab/tPLL_CROPseq. The tool get_barcodes.py was applied to extract the number copies of each gRNA barcode in each cell. Cells were assigned to gRNA barcodes if the most abundant barcode had UMI count >10 and >3-fold higher UMI count than the sum of all other barcodes detected in the same cell. Cells that did not meet these criteria were discarded as likely empty droplets or doublets.