Aggregate of Approximately 1.2 Million Human CRISPRi K562 Cells, Multiplexed Samples, 16 Probe Barcodes

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 fixed for 24 hours at 4°C following the demonstrated protocol Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling (CG000782).

Gene Expression and matching CRISPR libraries were generated as described in the GEM-X Flex Gene Expression Reagent Kit for Multiplex samples (CG000787) with the workflow modifications described in the Knowledge Base article What workflow modifications are required when performing CRISPR Screening with the Single Cell Gene Expression Flex assay?

The fixed cells were run as a 16-sample multiplexed experiment, where the sample was evenly split across 16 separate hybridization reactions, each using a unique Probe Barcode (sample sub-pooling). Custom probes designed to capture the sgRNAs and their gene targets were pooled and spiked in per the Custom Probe Design for Visium Spatial Gene Expression and Chromium Single Cell Gene Expression Flex Technical Note (CG000621). After hybridization, cells from each hybridization were pooled in equal proportions, washed, and then run across four GEM lanes. Note: a final cell concentration of approximately 14,000 cells/uL or higher is needed to target 320,000 cells. The 16-plex libraries were pooled and sequenced on an Illumina NovaSeq X at a read depth of 10,000 reads per cell for Gene Expression libraries and 5,000 reads per cell for CRISPR libraries.

Paired-end, dual indexing libraries were sequenced using the following scheme:

• 28 cycles Read 1
• 10 cycles i7
• 10 cycles i5
• 90 cycles Read 2

This dataset is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. 10x citation guidelines available here.