title: GSM4769378: Rep2_Cy5high; Homo sapiens; OTHER
library_construction_protocol: gDNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen), including RNAse A treatment. gDNA was quantified by TapeStation 2200 (Agilent), and then fragmented by sonication (either Q800R2 Sonicator (Qsonica) or Bioruptor Plus (Diagenode)) to 800-1000bp fragments. After gDNA was denatured, 10% of biotinylated probes (by mass; e.g. 10 μg probes for 100 μg gDNA)  were pre-coupled to Streptavidin beads (ThermoFisher) using the manufacturer recommended protocol, and were then hybridized to vector-containing gDNA fragments in 1× LiCl/Urea Buffer (the final 1× Buffer contains 25 mM Tris pH 7.4, 5 mM EDTA, 400 mM LiCl, 0.1% NP40, 0.1% SDS, 0.1% sodium deoxycholate, 1M urea) for 3 hours at 60ºC. After hybridization, beads were rinsed with 1× LiCl/Urea Buffer at 45ºC for 2-3 minutes. sgRNA-containing gDNA fragments were eluted by digesting RNA probes with Ambion® RNase Cocktail™ (ThermoFisher) and by degrading RNA with NaOH (Sigma). gDNA fragments were purified using the DNA Clean & Concentrator-5 kit (Zymo Research). Purified gDNA fragments were then PCR amplified (NEBNext Ultra II Q5, NEB) with FLI1F and FLIR primers in PCR1 (denaturation at 98ºC for 30 seconds, followed by 6-9 cycles of denaturation at 98ºC for 12 seconds, annealing at 69ºC for 60 seconds, and extension at 72ºC for 30 seconds, followed by a final elongation at 72ºC for 60 seconds). PCR1 products were purified using AMPure XP beads at 1.6× ratio by volume and manufacturer protocol. After purification, PCR2 was performed with NEBNext Ultra II Q5 (NEB) using standard Illumina indexed primers (e.g. D501 and D701) as follows: denaturation at 98ºC for 30 seconds, followed by 7-8 cycles of denaturation at 98ºC for 10 seconds and annealing and elongation at 72ºC for 40 seconds, followed by a final elongation at 72ºC for 60 seconds). PCR2 products were purified using AMPure XP beads at 1.3× ratio by volume and manufacturer protocol. Libraries were quantified by Tapestation 2200 (Agilent) and sequenced on the HiSeq 2500 or 4000 platforms (Illumina). As these libraries contain identical sequences upstream of the variable sgRNA region, pools submitted for sequencing contained at most 20% FLI-seq libraries, with the remainder of the pool containing high-diversity samples (e.g. standard RNA-seq). Other: pooled CRISPR
attributes.tag: GEO Accession
attributes.value: GSM4769378