The human K562 cell line, stably expressing KRAB-dCas9, was transduced with a pooled lentiviral CRISPRi lncRNA and protein-coding library containing approximately 6,903 sgRNAs. The CRISPRi library consisted of 4,045 sgRNAs, including those targeting 280 lncRNAs, 567 protein-coding genes, and 177 non-targeting sgRNA controls. The target genes for the remaining 2,681 sgRNAs were unknown and set to "ignore."
Transduced cells were allowed to recover for 48 hours, followed by 4 days of culture in selection media. The CRISPRi screen was stopped on day 6 and the selected cells were cryopreserved.
Cryopreserved cells were thawed, counted, and loaded onto the Chromium X.
Libraries were generated as described in the Chromium GEM-X Single Cell 5' Reagent Kits v3 with Feature Barcode technology for CRISPR Screening User Guide (CG000735). Libraries were sequenced on an Illumina NovaSeq 6000 to a read depth of approximately 20,000 mean reads per cell for Gene Expression libraries and 5,000 mean reads per cell for CRISPR screening libraries.
Paired-end, dual indexing sequencing scheme:
- 28 cycles Read 1
- 10 cycles i7
- 10 cycles i5
- 90 cycles Read 2