GSM6739091: eHAP FNLS + sgRNA SF3B3_55, no treatment; Homo sapiens; OTHER
Genomic DNA was extracted using QuickExtractTM DNA Extraction Solution (Lucigen #QE09050) according to the manufacturer's protocol. 1 µl (qsp. >200 cells) and was used for target site amplification using a 2-step PCR. In addition, each reaction contained 10 µl NEBnext Ultra II Q5 master mix as well as 1 µM of each forward and reverse primer. Primer pairs for PCR 1 were selected such that forward annealing primer is not closer than 7 nt to editing window position 1  but still allowing that 75 sequencing cycles will read the sequence. Primers were verified to anneal to a single position within the genome using BLAT11 and tested before use. Primers for PCR 1 (12 cycles) were flanked for the forward primer by: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' and for the reverse primer by 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'. 1.5 µl of PCR 1 were used as template for PCR 2 (14 cycles) and used forward primers 5′-AATGATACGGCGACCACCGAGATCTACAC-NNNNNNNN-ACACTCTTTCCCTACACGAC-3' (compatible to Illumina i5) and reverse primers 5′-CAAGCAGAAGACGGCATACGAGAT- NNNNNNNN- GTGACTGGAGTTCAGACGTG-3' (compatible to Illumina i7), with both containing 8N barcodes for multiplexing.  Primers were removed after the second PCR step with AMPure XP Reagent (Beckman #A63882). Library was quantified (QuantiFluor® dsDNA System; Promega #E2670) before being pooled (10 fmol per sample) for each time point & condition, run on 8% TBE-PAGE gel, then size selected for a range of 250-500 bp. Pooled libraries were then run on Bioanalyzer High Sensitivity DNA Kit, Agilent #5067-4626) before final pooling and subsequent run on a MiniSeq Sequencing System using the Miniseq High Output Kit (75 cycles) (Illumina #FC-420-1001) with a 10% of phiX spike-in.