Truseq sbs kit v3 chemistry

RNA was extracted using the Qiagen Qiashredder kit (79654) and the Qiagen RNeasy Mini Kit (74104) as per the manufacturer’s instructions (Qiagen Inc., Valencia, CA, USA). The RNA was DNase treated following the protocol in the RNeasy Mini Kit with Qiagen RNase-free DNase I (79254). Indexed PolyA libraries were prepared using 1 μg of total RNA and 13 cycles of amplification in the NEB Next Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Cat No: E7420S). RNA Integrity Values scores were >9 for all samples. Libraries were quantified by qPCR using a Kapa Library Quantification Kit for Illumina sequencing platforms (Kapa Biosystems, Wilmington, MA, USA, Cat No: KK4835). Pooled libraries were clustered at 15 pmol/l on the cBot and 2×100 bp sequencing was carried out using the High Throughput mode of a HiSeq 2500 using TruSeq SBS Kit v3 chemistry (Illumina)

Genomic DNA (gDNA) libraries were prepared from FF-derived DNA using the Agilent Sure Select^xt Target Enrichment System Automation Protocol (Agilent Technologies, Tokyo, Japan), and gDNA library preparation was done according to the manufacturer’s instructions, using 200ng DNA. gDNA libraries were prepared from FFPE-derived DNA using Agilent Haloplex^xt Target Enrichment System Automation Protocol (Agilent Technologies, Tokyo, Japan). The size distributions of the gDNA libraries were estimated by on-chip electrophoresis (High-Sensitivity DNA chips and High-Sensitivity D1000 Screen Tape) of a 1 µL sample on an Agilent 2100 Bioanalyzer/Agilent 2200 Tapestation (Agilent Technologies, Tokyo, Japan). The exome or target genes captured gDNA libraries were combined into 2 nM pooled stocks, denatured and diluted to 10 pM with pre-chilled hybridization buffer and loaded into TruSeq PE v3 flowcells on an Illumina cBot, followed by indexed paired-end sequencing (101 + 7 + 101 bp) on a Illumina HiSeq 2500 using TruSeq SBS Kit v3 chemistry (Illumina, San Diego, California, USA).