Truseq pcr free sample preparation kit

Genomic DNA was extracted from livers of GI mice using a Qiagen DNeasy blood and tissue kit. DNAs were further cleaned by phenol and chloroform to meet quality requirements for genome sequencing. Genome sequencing was completed in the University of Wisconsin – Madison Biotechnology Center. DNA concentration and sizing were verified using the Qubit dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA, USA) and the Agilent DNA 1000 chip (Agilent Technologies, Inc., Santa Clara, CA, USA), respectively. Samples were then prepared using the TruSeq PCR Free Sample Preparation kit (Illumina Inc., San Diego, CA, USA), with minor modifications. Libraries were size-selected for an average insert size of 550 bp using SPRI-based bead selection. Quality of the finished libraries was assessed using an Agilent DNA 1000 chip and qPCR quantification was performed using the Kapa Illumina NGS Library Quantification Kit (KAPA Biosystems, Wilmington, MA). Libraries were standardized to a concentration of 2 nM. Cluster generation was performed using the Illumina Rapid PE Cluster Kits v2 and the Illumina cBot. Paired-end, 100-bp sequences were generated using Rapid v2 SBS chemistry on an Illumina HiSeq2500 sequencer. Images were analyzed using the standard Illumina Pipeline, version 1.8.2.

Isolated mutant populations were singly cloned by limiting dilution followed by expansion in HFFs. Genomic DNA from three mutant strains and a wild-type strain was isolated using genomic DNA Clean & Concentrator (Zymo Research).
For the DNA library preparations, 1 µg of gDNA was first sheared down to 200 to 300 bp using the Covaris S2 per the manufacturer’s recommendations. Paired-end sequencing libraries were prepared using Illumina’s TruSeq PCR free sample preparation kit. The target insert size of 200 to 250 bp was size selected using SPRI Ampure XP purification. Following DNA library construction, library size distribution was checked using the Agilent Bioanalyzer high-sensitivity assay. Library quantification was done via qPCR (Stratagene MX3005P). DNA libraries were sequenced using the Illumina HiSeq 2000 in one lane on a flow cell with sequencing paired-end read length at 2 × 50 bp. Reads were demultiplexed using CASAVA (version 1.8.2).