CRISPR screen and single-cell sequencing project: Difference between revisions
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#Take the top gRNA for each gene, and order primers for the gRNA ([[File:validation_primers.txt]]) with the m scaffold only (for now). | #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 | #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 sequencing | #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. |
Revision as of 00:34, 6 May 2015
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.