CRISPR screen and single-cell sequencing project
Jump to navigation
Jump to search
The goal of this project is to screen H9-GFP tagged hESCs with 4 curated libraries and then use single-cell sequencing to determine which gRNA was incorporated into which cell and gene expression in each cell. The four libraries and some annotation info is listed in this file (File:Crispr screen single cell sequencing library.xlsx).
Library Design:
- From the curated gene lists, take all spacers for each gene from the gRNA list.
- Check each spacer for multi-mapping and that each spacer maps to the correct location.
- Keep only the top 3 spacers for each gene.
- Include 3 negative controls in each library, barcode using bc_25mer barcodes.
- Order gChip. See 05/05/2015 Library Order.
- Use CRISPR Library Prep Protocol to prepare library.
Experimental Plan:
- Grow H9-GFP cells for 2 weeks.
- Run single cell sequencing on cells
Validation Assay:
- Pick two genes from each library for validation (File:Validation library.txt)
- Take the top gRNA for each gene, and order primers for the gRNA (File:Validation primers.txt) with the m scaffold only (for now).
- PCR to synthesize gRNAs, and regular CRISPR Library Prep Protocol to put gRNAs into bacteria
- Incorporate gRNAs into bulk H9-GFP tagged cells (one gRNA per batch of cells). Perform bulk RNA sequencing with 3 technical replicates for each gRNA, 3 negative control gRNAs resulting in 27 sequencing runs in total.
- The goal of this validation assay is to determine what sequencing depth we need to detect the gRNA, how well we can detect the gRNA, and gRNA efficiency with the modified scaffold.