GSM9395646: SW1 CRISPR KO library metastasis; Mus musculus; OTHER
DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, 69504). gDNA equivalent of 500x library coverage was amplified using Hifi high fidelity polymerase (Stellar Scientific, RAD HF1100), and primers synthesized in IDT according to previously described sequences (DOI: 10.1038/nprot.2017.016). Each PCR reaction contained 500 ng gDNA, 0.5 μL HiFi high fidelity polymerase, 10 μL of 5x buffer, 2 μL of forward and reverse primers and molecular biology grade water up to 50 μL. A total of 8000 ng was amplified for each sample. PCR conditions were as follows: 1 min at 95 °C, followed by 15 sec at 95 °C, 15 sec at 65 °C, 30 sec at 72 °C repeated for 25 cycles and finally 5 min at 72 °C. Finally, PCR products were purified with Agencourt AMPure XP SPRI beads according to the manufacturer's instructions (Beckman Coulter, A63880). Product purity was evaluated using Agilent 2100 BioAnalyzer and DNA 1000 Kit (Agilent Technologies, 5067-1504), and concentration was measured with Qubit™ 1X dsDNA HS Assay (Qubit, Q33230). Lastly, the qualified RNA-seq libraries are sequenced using Illumina NovaSeq6000, to get on average 1.5 million, single-end 61 bp reads to achieve >500X coverage.