A competitive precision CRISPR method to identify the fitness effects of transcription factor binding sites
for read count ratios for cell lineages edited with repair templates harboring only the original E-box sequence as well as pairs of cell lineages edited with mutated and original sequence and the similar flanking mutations are shown for sequence tags with two flanking mutations for PAICS, SHMT2 and PPAT and sequence tags with one flanking mutation for RPL23 and HK2.
All immunoprecipitated DNA isolated from transfected cells was amplified for 30 cycles in two reactions using similar PCR1 conditions and primers as described above for gDNA samples. In addition, 10 μg of input DNA from each transfected condition was amplified in four parallel reactions. PCR1 products were purified using 1.5× AMPure XP beads, and 20% of purified DNA was used as a template in PCR2 for eight cycles with Illumina primers as above. Final libraries were purified using 0.
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