2.6. DNA Exon-Seq Analysis

For the generation of standard exome capture libraries, the Agilent SureSelect Target Enrichment protocol for the Illumina paired-end sequencing library (ver. B.3, June 2015) was used with 1 µg input gDNA. In all cases, the SureSelect Human All Exon V6 or SureSelect Mouse All Exon probe set was used. DNA quantification and DNA quality were analyzed using PicoGreen and agarose gel electrophoresis. One microgram of genomic DNA from each cell line was diluted in EB buffer and sheared to a target peak size of 150–200 bp using the Covaris LE220 focused ultrasonicator (Covaris, Woburn, MA, USA) according to the manufacturer’s recommendations. The 8 microTUBE Strip was loaded into the tube holder of the ultrasonicator, and the DNA was sheared using the following settings: mode, frequency sweeping; duty cycle, 10%; intensity, 5; cycles per burst, 200; duration, 60 sec × 6 cycles; and temperature, 4–7 °C. The fragmented DNA was repaired, an ‘A’ was ligated to the 3′ end, and Agilent adapters were then ligated to the fragments. Once ligation was assessed, the adapter-ligated product was PCR amplified. The final purified product was quantified using the TapeStation DNA screentape D1000 (Agilent, Santa Clara, CA, USA). For exome capture, 250 ng of the DNA library was mixed with hybridization buffers, blocking mixes, RNase block, and 5 µL of SureSelect all-exon capture library, according to the standard Agilent SureSelect Target Enrichment protocol. Hybridization to the capture baits was performed at 65 °C using the heated thermal cycler lid option at 105 °C for 24 h on a PCR machine. The captured DNA was then washed and amplified. The final purified product was quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms), analyzed using the TapeStation DNA screentape D1000 (Agilent), and sequenced using the HiSeq™ 2500 platform (Illumina, San Diego, CA, USA).