NextSeq 500 sequencing; In vivo CRISPR/Cas9 screen to identify candidate therapeutic gene targets to enhance docetaxel efficacy in prostate cancer
In vivo CRISPR/Cas9 screen to identify candidate therapeutic gene targets to enhance docetaxel efficacy in prostate cancer
DNA was extracted from each whole tumour using the Blood and Cell Culture DNA Maxi kit (Qiagen) according to the manufacturer's instructions. DNA was extracted from 100mg of ground tumour (or entire tumour if weight under 100mg) using QIAamp DNA Mini kit (Qiagen) as per the manufacturer's instructions. DNA was prepared for deep sequencing by conducting a two-step PCR. The initial PCR amplified a region of the gRNA cassette to maintain library representation, while the second PCR added the primers required for sequencing. PCR was repeated 35 times for 100 fold library representation. All PCR products per sample were combined and used in the second PCR round to add the primers required for sequencing. PCR products from each sample were again combined and concentrated using a QIAquick PCR Purification Kit (Qiagen) as per the manufacturer's instructions. Each sample was run on a 1.5% agarose gel. Bands were excised and DNA purified using a QIAquick Gel Extraction Kit before sending for sequencing.