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 1: Line 1:
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 [[File:Crispr_screen_single_cell_sequencing_library.xlsx]].
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:
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.

Revision as of 00:23, 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.