Genome-wide allele screen and chromosome knockout project
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Library Design Protocol:
Dry Lab
- Input data is either phased haplotypes (in HapCut output format: File:Haplotype.chr21.combine.txt) or SNP Calls in tab delimited format (File:Pgp1 snp calls.tsv, File:HapmapSnpsCEU.txt).
- Filter SNPs for snps that fall in PAM motifs (GG or CC) motifs
- Construct 23 bp spacer sequences + PAM motifs from these SNPs
- Using bowtie, check spacer sequences for multi-mapping. Ensure each spacer + PAM sequence is 2 or 3 mismatches away from any other location on the genome.
- Check SNP calls or Haploytping data for quality
- Barcode each individual library from the bc_25mer set (
Wet Lab