Targeted deep sequencing of Mus musculus: Rrm1:+/Y285A colon
First, the qualified genomic DNA sample was randomly fragmented into fragments with a base pair peak of 150 to 200bp, and then adapters were ligated to both ends of the resulting fragments. The adapter-ligated templates were purified by the Agencourt AMPure SPRI beads and fragments with insert size about 200bp were excised. Extracted DNA was amplified by ligation-mediated polymerase chain reaction (LM-PCR), purified, and hybridized to the SureSelect Biotinylated RNA Library (BAITS) for enrichment. Hybridized fragments were bound to the strepavidin beads whereas non-hybridized fragments were washed out after 24h. Captured LM-PCR products were subjected to Agilent 2100 Bioanalyzer to estimate the magnitude of enrichment. Each captured library was then loaded on Hiseq4000 or DNBseq platform, and we performed high-throughput sequencing for each captured library independently to ensure that each sample meet the desired average fold-coverage. Raw image files were processed by Illumina base calling Software 1.7 for base calling with default parameters and the sequences of each individual were generated as 90bp paired-end reads.