Lb carbenicillin plates

The gRNA library targeting murine chromatin regulators was constructed as previously described.^{15 (link)} Briefly, it consisted of sgRNA sequences (six per gene) targeting 612 mouse chromatin regulators that were designed using BROAD sgRNA Designer^{75 (link)} and 36 non-targeting control sgRNAs.^{15 (link)}This library was synthesized by Agilent Technologies and cloned into the pUSEPR lentiviral vector to ensure a library representation of >10,000X using a modified version of a previously described protocol.^{75 (link)} Then, it was selectively amplified using barcoded forward and reverse primers that append cloning adapters at the 5 - and 3 -ends of the sgRNA insert, purified using the QIAquick PCR Purification Kit (Qiagen), and ligated into BsmBI-digested and dephosphorylated pUSEPR vector using high-concentration T4 DNA ligase (NEB). Ligated pUSEPR plasmid DNA was electroporated into Endura electrocompetent cells (Lucigen), recovered for 1 h at 37°C, plated across four 15cm LB-Carbenicillin plates (Teknova), and incubated at 37°C for 16 h. The total number of bacterial colonies was quantified to ensure a library representation of >10,000X. Bacterial colonies were scraped and briefly expanded for 4 h at 37°C in 500mL of LB-Carbenicillin. Plasmid DNA was isolated using the Plasmid Plus Maxi Kit (Qiagen).