scRNA-seq of gRNA-derived oligos from MDA-MB-231 with RBP perturbations using short-read transcriptomics
CRISPore-seq: Library preparation and sequencingPooled libraries were transduced into MDA-MB-231-CRISPRi cells, and, at 24 hours after transduction, cells were lifted and washed in PBS before being re-plated in puromycin-containing selection media. The survival was below 4% after 2 days of selection in 2 g/mL puromycin (Invivogen ant-pr-1). On day 7 post-transduction, the cells were collected and processed for cell barcoding and transcriptome capture.We performed cell-hashing and barcoding for ECCITE-seq47 and separated cells into 8 sub-pools. For each pool, we resuspended 1 million cells in 100 l staining buffer (2% BSA, 0.01% Tween in PBS). We then added 10 l Human TruStain FcX Receptor Blocking Solution (BioLegend) and incubated it on ice for 10 minutes. We then added 0.5 g hashing antibodies to each of the 8 sub-pools (BioLegend), incubated on ice for 30 minutes and washed cells 3 times with 1 ml staining buffer. Cells were spun down at 4C for 5 minutes at 450 g. Cells were resuspended in 50 l ice cold PBS each and 1 l was used to determine cell count. Cells from all 8 sub-pools were merged at equal proportions in 1 ml staining buffer, spun down at 350 g for 5 min at 4C. Cells were resuspended in PBS at 1500 cell/l and filtered through a 40 m strainer (Flowmi).Using the Chromium Single Cell Immune Profiling Solution v1.0 kit (10x Genomics), we targeted recovery of 12,000 cells per lane (superloading). We followed the 10x Genomics single cell assay with the following modifications: 5.9 l of 2 M gd_RT_V4 primer was added to the RT reaction and 1 l of 1 M of hashtag & guide-tag additive were added to the cDNA PCR as additives.Post GEM-RT cleanup and cDNA amplification was carried out as described in the 10x Genomics single cell kit. Following cDNA library amplification, we set aside 300 ng of full-length cDNA library at 4 C for long-read sequencing. A total of 100 ng of cDNA was used to process the 5 Gene Expression (GEX) library using fragmentation, end repair and A-tailing. Hashtag and guide-tag library preparations were performed as in CITE-seq48. Hashtag and guide RNA libraries were separated from cDNA libraries using 0.6 SPRI as desribed in Stoeckius et al. 2017 prior to fragmentation. Gene expression (GEX), hashtag and guide RNA libraries were quantified and diluted to 4 nmol and pooled in the following ratio: 5% Hashtag library, 10% Guide RNA library, 83% Gene expression library and 2% PhiX spike in. Each library was sequenced on a NextSeq550 75-cycle high-output run (Illumina)."
SUB15785275
CRISPore-seq_short-read_GDO