title: NSD3-short is a multifunctional adaptor protein that couples BRD4 to the CHD8 chromatin remodeler
design: Design and construction of pooled sgRNA library   For saturating mutagenesis of Chd8 genes, sgRNAs were designed to target all possible PAM NGG sequence on the plus or minus strand of the protein-coding region (sgRNAs with sever off-target cutting predictions were filtered out to minimize the false positive phenotypes). Single strand sgRNAs were synthesized through array platform (Customarray), PCR amplified and then Gibson cloning into Bsmb1-digested LRG sgRNA lentiviral expression vector (#65656; Addgene). The Gibson ligation product was transformed to eletrocompetent cells (#C6400-3, Invitrogen) to ensure at least 300x library coverage of each sgRNA designs. The overall quality of the pooled sgRNA library was measured through deep sequencing (data not showed).    CRISPR/Cas9-mediated saturating mutagenesis of candidate genes  Lentivirus of pooled sgRNAs targeting Chd8 were produced as described above and the viral titer was measured through a serial dilutions. To ensure that a single sgRNA was transduced per cell, the viral volume for infection was chosen to achieve a multiplicity of infection (MOI) of 0.3–0.4. To maintain the representation of each sgRNA, at least 1000 leukemia cells transduced with individual sgRNAs were maintained throughout the whole negative selection screening experiment time course. The genomic DNA was extracted at the indicated time points with QiAamp DNA mini kit (#51304; Qiagen), following the manufacturer’s instructions. The pooled screening illumine deep sequencing libraries were constructed as described previously (Shi et al., 2015). Briefly, multiple independent PCR reactions were set up to amplify the sgRNA cassette to maintain at least 500× sgRNA library representation using the 2X Phusion Master Mix (#F-548; Thermo Scientific). PCR products were pooled and subjected to Illumina MiSeq library construction and sequencing.