gRNA_pooled_screen
The lysine methyltransferase U6-sgRNA-EFS-GFP library was pooled at equimolar ratio and used to generate a lentiviral supernatant as described above. A dilutions series of this virus correlated with GFP positivity in infected cells to derive the viral titer multiplicity of infection (MOI). The total number of RN2c cells was chosen to achieve at least 500-fold representation of each sgRNA in the initially infected cell population. To ensure that a single sgRNA was transduced per cell, the viral volume for infection was chose to achieve an MOI of 0.3-0.4. Genomic DNA was extracted at the indicated time points using QiAamp DNA mini kit (Qiagen #51304), following the manufacturer’s instructions. To maintain >500x sgRNA library representation, 16-20 independent PCR reactions were used to amplify the sgRNA cassette, which were amplified for 20 cycles with 100 ng of starting gDNA using the 2X Phusion Master Mix (Thermo Scientific #F-548). PCR products were pooled and subjected to illumina MiSeq library construction and sequencing. 100-200 ng gDNA was amplified for 20 cycles with 2X Phusion Master Mix (Thermo Scientific #F-548) following the manufacture’s instructions. The PCR clean up products were subjected to illumine sequencing library construction. Briefly, the PCR product was end repaired with T4 DNA polymerase (NEB), DNA polymerase I (NEB), and T4 polynucleotide kinase (NEB). An A overhang was added to the end-repaired DNA using Klenow DNA Pol Exo- (NEB). The DNA fragment was then ligated with diversity-increased barcoded Illumina adaptors followed by 5 pre-capture PCR cycles. Barcoded libraries were pooled at equal molar ratio and subjected to massively parallel sequencing through a Mi-Seq instrument (illumina) using paired-end 150 bp sequencing (MiSeq Reagent Kit v2; Illumina MS-102-2002).