Amplicon seq of HAP1 (dG2M population, STLC)
Cells expressing S.p. dCas9-VP64-p65-Rta (dCas9-VPR) were infected with the Human CRISPR Activation Pooled Library (Calabrese P65-HSF) (Sanson et al., Nat Commun. 2018, PMID: 30575746) at an MOI of 0.2 and 200x coverage. After puromycin selection, cells were cultured with S-trityl-L-cysteine (STLC), an Eg5 inhibitor, for 6 days and then fixed with 70% cold ethanol. The cell population with DNA content of 4C (corresponding to diploid G2M) was purified by flow cytometry, and genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions. The extracted genomic DNA was then subjected to gRNA amplification following the protocol from Olivieri and Durocher, STAR Protoc. 2021 (PMID: 33598657).