title: GSM4649314: DMSO_rep1; Homo sapiens; OTHER
library_construction_protocol: Cells were lysed in 10 mM Tris-HCl, pH 8.0, 100 mM EDTA, 0.5% SDS, 200 μg/ml RNase A and incubated at 37 °C for 1 hour followed by the addition of 1mg/ml of proteinase K for an overnight incubation at 50 °C. The resulting lysates were extracted three times with phenol solution equilibrated with 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (Sigma P4557) mixed with chloroform (1:1 v/v) using tubes with phase lock gel (Tiangen WM5-2302831). The aqueous phase was then mixed with 0.2 volume of 10 M ammonium acetate and 1 volume of isopropanol, resulting the immediate formation of cloudy DNA precipitates. Precipitated DNA was then transferred by a pipet tip to a tube containing 75% (v/v) ethanol. This process was repeated twice to ensure complete removal of residual organic solvents. Afterwards, genomic DNA was dissolved in 1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA by incubation at 50 °C for three hours. DNA fragments containing sgRNA sequences were amplified from isolated genomic DNA by two rounds of PCR using NEBNext Ultra II Q5 master mix (NEB). For the first round of PCR, forty-eight 50-μL PCR reactions (each containing 2 μg of genomic DNA template) were performed with the forward primer NGS-Lib-KO-Fwd-1 (5'- CCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNGCTTTATATATCTTGTGGAAAGGACGAAACACC -3') and the reverse primer NGS-Lib-KO-Rev-0 (5'- CAGACGTGTGCTCTTCCGATCTCCGACTCGGTGCCACTTTTTCAA-3') with a thermal cycler program consisting of initial denaturation at 98°C for 5 minutes, followed by 16 cycles of (98°C denaturation for 10 seconds, 69°C annealing for 30 seconds, and 65°C extension for 45 seconds), and a final extension at 65°C for 5 minutes. Products of the firstround PCR were pooled and purified by a DNA clean and concentrator kit (Zymo) and diluted to 2 ng/μL. For the second round of PCR, six 50-μL PCR reactions (each containing 2 ng of the purified first-round PCR product) were performed for each sample with the forward primer NGS-Lib-KO-Fwd-2 (5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') and one of four indexed reverse primers (NGS-Lib-KO-Rev-1, 2, 3, 4) (5'-CAAGCAGAAGACGGCATACGAGAT[8-nucleotide index]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'). The same cycling conditions were used for second-round PCR as the first-round PCR except 18 cycles were used. Products of the second-round PCR reactions were subjected to electrophoresis on a 2% agarose gel. The expected ~300 bp amplicons were excised and extracted from the gel and sequenced by Illumina HiSeq PE150 (Novogene).
attributes.tag: GEO Accession
attributes.value: GSM4649314