30k A549, Lung Carcinoma Cells, Treatments Transduced with a CRISPR Pool, Multiplexed, 6 CMOs

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. Cells that received five separate drug treatments (DZNep, Trichostatin A, Valproic Acid, Kinetin, Resveratrol) and cells with no treatment (the same cells used to generate the non-multiplexed sample) were multiplexed at equal proportions with 1 CMO per sample type, resulting in a pooled sample labeled with 6 CMOs.

Libraries were prepared following the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for CRISPR Screening and Cell Multiplexing User Guide (CG000389) and sequenced on Illumina NovaSeq 6000.

Single Cell 3’ Cell Multiplexing v3.1 Dual Index Library

• Sequencing Depth: 69,736 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)

Pooled Multiplexed Sample - Key Metrics

• Estimated Number of Cells: 19,126
• Cells Assigned to a Sample: 15,285

Individual Multiplexed Samples - Key Metrics