title: GSM8798089: Part1; Homo sapiens; OTHER
library_name: GSM8798089
library_construction_protocol: Cell samples were lysed in 1 ml DNAzol by vigorous pipetting according to the manufacture's protocol (Molecular Research Center inc.).  DNA was precipitated in 0.5 ml 200 proof ethanol (VWR).  30 to 50 mg of tumor samples were lysed in 5 ml DNAzol by a motorized tissue homogenizer (Kinematica AG PT1200), centrifuged at 3,200 g for 15 minutes at 4 °C, and transferred to a new tube.  gDNA was then precipitated by 2.5 ml 200 proof ethanol.  Precipitated DNA was pelleted by centrifugation at 20,000 g, washed twice in 75% ethanol.  gDNA was solubilized in 0.5 ml or 1.0 ml of water for cell and tumor samples, respectively To reduce PCR bias, amplification of sgRNA sequences from each sample gDNA was performed in triplicate and pooled at each step.  First round of PCR amplification was performed using 50 to 100 µg gDNA, Hotstart Taq (Takara), and 1 µM CRISPR F1 and CRISPR R1 primers (IDT). CRISPR F1 - AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG CRISPR R1 - TCTACTATTCTTTCCCCTGCACTGTtgtgggcgatgtgcgctctg Thermal cycles consisted of 95 °C for 5 minutes, followed by 18 cycles of 95 °C for 30 seconds, 60 °C for 30 seconds, 72 °C for 30 seconds, and a final cycle of 72 °C for 5 minutes.  Amplicons from the same samples were pooled and a second round of PCR amplification was performed to add sample barcode and Illumina adaptor sequences using 20 µl of PCR1 and 0.5 µM  CRISPR R2 and barcoded forward primers. See Supplemental Data 2 for barcode information. CRISPR R2 - CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtctactattctttcccctgcactgt Thermal cycles consisted of 95 °C for 5 minutes, followed by 20 cycles of 95 °C for 15 seconds, 58 °C for 30 seconds, 72 °C for 30 seconds, and a final cycle of 72 °C for 5 minutes.  Amplicons from the second round of PCR were pooled at equal concentrations, were purified using Ampure XP Beads according to the manufacture's protocol (Beckman Coulter).  Purified amplicons were subjected to Illumina NexSeq 500 75bp single end sequencing.
identifiers.namespace: GEO
identifiers.id: GSM8798089_r1