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To assess the performance of CRISPR Detect, cells were prepared to express just one guide RNA (sgRNA). Three guide RNAs were independently cloned into CROP-seq-Opti-eGFP and packaged into lentiviral particles. LN18 cells were separately transduced with the three lentiviral vectors at an MOI of 0.5. After 3 days of growth, GFP+ transduced cells were selected with FACS and pooled at an equal ratio. This experimental design examines the performance of the assay, by reducing background from biological variation (Figure 1).
After fixation with Evercode Cell Fixation v2, cells were processed with Evercode WT Mega v2 and CRISPR Detect. Whole transcriptome and CRISPR sgRNA libraries from one sublibrary were sequenced together on an Illumina Nextseq 550 with a High Output flow cell. The data was analyzed with the Parse Biosciences Analysis Pipeline.
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At a sequencing depth of 3,000 reads per cell in the CRISPR Detect library, the library reached 98% sequencing saturation. The results reveal high overall sgRNA detection rates with 78% of cells having a single sgRNA as expected (Figure 2).
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Learn More About CRISPR Detect
CRISPR Results Experimental Summary Report (HTML) Digital Gene Expression (DGE) Matrix (500 KB) All Gene (CSV) Cell Metadata (CSV) Guide Assignment (CSV)
Whole Transcriptome Results Experimental Summary Report (HTML) Digital Gene Expression (DGE) Matrix (220 MB) All Gene (CSV) Cell Metadata (CSV)