10K A375 cells, a human cell line derived from malignant melanoma cells, were transduced with non-target and target sgRNA. A375 dCas9 Parental Cells were transfected separately with individual guides (either non-targeting or Rab1a sgRNA,with Capture Sequence 2 inserted into the hairpin structure of the sgRNA). Selected cells (cultured in a selection media) for each condition were individually frozen. Aliquots of Rab1a and non-target sgRNA containing cells are thawed, counted, and mixed 1:1. The same cells were used to generate Chromium Single Cell 3’ (Single Index) data.
Libraries were prepared following the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for CRISPR Screening (CG000316) and sequenced on Illumina NovaSeq 6000.
Single Cell 3’ CRISPR Screening v3.1 Dual Index Library
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Sequencing Depth: 60,435 read pairs per cell
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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)
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Analysis Parameters Used: Run with --expect-cells=10,000
Key metrics were:
- Cells detected: 11,791
- Median UMI counts per cell: 25,248
- Median genes per cell: 4,865