GSM9542785: LN229,CRISPRa,RM6,rep2; Homo sapiens; OTHER
Genomic DNA was purified from cell pellets using NucleoSpin Blood L gDNA extraction kits (Nacherey-Nagel # 740954) in accordance with the manufacturer's instructions and yields and purity were confirmed by nanodrop. Amplicons were generated by PCR to introduce sequencing adaptors and sample-specific indexes using a common forward primer within the U6 promoter and reverse primers which bind the sgRNA constant region downstream of the second sgRNA and contain the sample-specific indexes. PCR reactions comprised 2x NEB Next Ultra II Q5 polymerase (NEB # M0544), 10 µg of purified gDNA and 1 µl of each 100 µM primer per 100 μl reaction. PCR amplification was performed with the following cycling conditions: denaturation at 98ºC for 30 secs, 22 cycles of denaturation at 98ºC for 10 secs then annealing and extension at 67ºC for 75 secs, and a final extension at 72ºC for 5 mins. All gDNA was PCR amplified and all reactions for each sample were mixed post-amplification. 300 µl of amplified PCR was subject to a 0.5-0.65X double-sided SPRI selection to enrich for the amplicon and remove excess primer. Sample purity and concentration were determined using an Agilent Tapestation and used to calculate equimolar sample pooling. Pooled libraries were sequenced on a NextSeq550 or NovaSeq 6000 platform with paired end reads using custom read 1, read 2 and index read 1 primers and 10% PhiX achieving a minimum of 6 million reads per sample (i.e. ≥300 reads/library element).