Whole genome sequencing of mouse B10.RIII: 13 weeks female spleen
Illumina TruSeq Nano DNA Library Prep kit was used to prepare uniquely indexed paired-end (2x150bp) libraries of genomic DNA (gDNA). Briefly, the genomic DNA was fragmented to ~ 500 bp insert size on the Covaris which generates dsDNA fragments with 3' or 5' overhangs. The sheared DNA is blunt-ended and library size selection done using sample purification beads. A single 'A' nucleotide is added to the 3' ends of the blunt fragments to prevent them from ligating to each other during the adapter ligation reaction. A corresponding single 'T' nucleotide on the 3' end of the adapter provides a complementary overhang for ligating the adapter to the fragment. The indexed adapters are ligated to the ends of the DNA fragments and then PCR-amplified to enrich for fragments that have adapters on both ends. The final purified product is then quantitated by qPCR before cluster generation and sequencing.