A549 lung carcinoma cells that expressed dCas9-KRAB were transduced with a pool containing 93 total sgRNAs (90 sgRNAs targeting 45 different genes and 3 non-targeting control sgRNAs, all using Capture Sequence 2 inserted into the hairpin structure of the sgRNA). Cells were obtained by 10x Genomics from MilliporeSigma. Selected cells (cultured in a selection media) for each condition were individually frozen. Aliquots of cells were then thawed and counted. The same cells were used as part of the multiplexed sample.
Libraries were prepared following the Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with Feature Barcoding technology for CRISPR Screening User Guide (CG000316) and sequenced on Illumina NovaSeq 6000.
Single Cell 3’ CRISPR Screening v3.1 Dual Index Library
- Sequencing Depth: 21,401 read pairs per cell
- Paired-end, dual indexing Read 1: 28 cycles (16 bp barcode, 12 bp UMI); i5 index: 10 cycles (sample index); i7 index: 10 cycles (sample index); Read 2: 90 cycles (transcript)
Key Metrics
- Estimated Number of Cells: 5,867
- Median Genes per Cell: 3,194
- Median UMI Counts per Cell: 10,773