GSM7716952: HCC1143, CROPseq; Homo sapiens; RNA-Seq
For scRNA-seq of control samples, cells were seeded into 15cm circular plates and incubated for 72 hours. After the incubation, the cells had a confluence <50% and were dissociated and subjected to scRNA-seq (10X Genomics). For CROP-seq screening, sgRNA containing lentiviruses were transduced into Cas9 expressing breast cancer cell lines HCC1143 and HCC38 (in 15cm plate-format) in presence of 8ug/ml polybrene, at an estimated MOI < 0.2. After 24h, the lentivirus containing media was removed, cells were washed with PBS, and puromycin-containing media (3ug/ml) was added to the cells for 48-96h until all control cells (not virus-infected) were dead. The cells were grown for 14 days post sgRNA transduction to allow potential reprogramming effects to take place, before the scRNAseq (10X Genomics). For control samples, the resulting suspension of human tumor cells (HCC38, HCC1143 in batch 1, and MCF7, HCC2157 in batch 2) was adjusted to a concentration appropriate for loading into a 10X genomics Chromium controller with an expected output of 5000 cells/each cell line. Samples within each batch were pooled into the same well and libraries constructed according to the manufacturer's instructions. Libraries were sequenced on an Illumina Novaseq 6000, aiming for approximately 100,000 reads/cell. For CROPseq, the CRISPR perturbed cell lines (HCC38, HCC1143) were adjusted to a concentration appropriate for loading into a 10X genomics with an expected output of 10,000 cells/each cell line. The 10x libraries were prepared based on manufacturer's instructions with feature barcode CRISPR screening protocol. Libraries were sequenced on an Illumina Novaseq 6000, aiming for approximately 100,000 reads/cell.