title: GSM4333001: Cas9, 2; Homo sapiens; RNA-Seq
library_construction_protocol: For each replicate, genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen), and crRNA barcodes were amplified through nested PCR. For this, genomic DNA extracts were split into portions of 2 µg and diluted with ddH2O to a volume of 67 µL, and mixed with 33 µL of PCR#1 master mix (20 µL Q5-reaction buffer (NEB), 2 µL 10 mM dNTPs (NEB), 5 µL 10 µM primer F1 (Shalem et al., 2014), 5 µL 10 µM primer R1 (Shalem et al., 2014), 1 µL Q5 (NEB)). PCR#1 was run at 70˚C annealing temperature for 18 cycles. After completion, all reactions were pooled, and 5 µL of the PCR#1 pool were diluted with 62 µL ddH2O, and mixed with 33 µL PCR#2 master mix (20 µL Q5-reaction buffer (NEB), 2 µL 10 mM dNTPs (NEB), 5 µL 10 µM primer F0x (Shalem et al., 2014), 5 µL 10 µM primer R0y (Shalem et al., 2014), 1 µL Q5 (NEB)). PCR#2 was run at 70˚C annealing temperature for 24 cycles. PCR products were gel-purified (QIAquick Gel Extraction kit, Qiagen) and submitted for sequencing on a HiSeq 4000 (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM4333001