CRISPR screen and single-cell sequencing project: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>YanWu
No edit summary
>YanWu
No edit summary
Line 17: Line 17:
#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 (3 technical replicates for each gRNA, 3 negative control gRNAs, 27 gRNAs total).
#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:

  1. From the curated gene lists, take all spacers for each gene from the gRNA list.
  2. Check each spacer for multi-mapping and that each spacer maps to the correct location.
  3. Keep only the top 3 spacers for each gene.
  4. Include 3 negative controls in each library, barcode using bc_25mer barcodes.
  5. Order gChip. See 05/05/2015 Library Order.
  6. Use CRISPR Library Prep Protocol to prepare library.

Experimental Plan:

  1. Grow H9-GFP cells for 2 weeks.
  2. Run single cell sequencing on cells

Validation Assay:

  1. Pick two genes from each library for validation (File:Validation library.txt)
  2. Take the top gRNA for each gene, and order primers for the gRNA (File:Validation primers.txt) with the m scaffold only (for now).
  3. PCR to synthesize gRNAs, and regular CRISPR Library Prep Protocol to put gRNAs into bacteria
  4. 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.