title: GSM3754729: Brunello-edited Nalm6 cells, S3_d0_short_term_rep2; Homo sapiens; OTHER
library_construction_protocol: At time of DNA extraction, 6 mL of NK Lysis Buffer (50 mM Tris, 50 mM EDTA, 1% SDS, pH 8) and 30μL of 20mg/mL Proteinase K were added to the frozen cell sample and incubated at 55°C overnight. The following day, 30μL of 10mg/mL RNase A, diluted in NK Lysis Buffer to 10mg/mL and then stored at 4°C, was added to the lysed sample, which was then inverted 25 times and incubated at 37°C for 30min. Samples were cooled on ice before addition of 2mL of pre-chilled 7.5M ammonium acetate to precipitate proteins. The samples were vortexed at high speed for 20s and then centrifuged at ≥ 4,000×g for 10min. After the spin, a tight pellet was visible in each tube and the supernatant was carefully decanted into a new 15mL tube. 6mL 100% isopropanol was added to the tube, inverted 50 times and centrifuged at ≥ 4,000×g for 10min. Genomic DNA (gDNA) was visible as a small white pellet in each tube. The supernatant was discarded, 6mL of freshly prepared 70% ethanol was added to the tube and inverted 10 times and centrifuged at ≥4,000×g for 1min. The supernatant was discarded by pouring; the tube was briefly spun, and remaining ethanol was removed using a pipette. After air drying for 10-30min, 500μL of 1x TE buffer was added, the tube was incubated at 65°C for 1h and at room temperature overnight to fully resuspend the DNA. The next day, the gDNA samples were vortexed briefly, and gDNA concentration was measured. Using all collected gDNA (1000x coverage) divided into 100µL PCR reactions with 5µg of DNA per reaction. We used Takara ExTaq DNA Polymerase and the default mix protocol with the following PCR program: (95° 2min, (98° 10sec, 60° 30sec, 72° 30sec) x 24, 72° 5min). PCR products were gel purified using the QiaQuick gel extraction kit (Qiagen). The purified, pooled library was then sequenced on a HiSeq4000 with ~5% PhiX added to the sequencing lane.
attributes.tag: GEO Accession
attributes.value: GSM3754729