Seqplex dna amplification kit

>500 ng of genomic DNA is sheared to ~300 bp long fragments prior to immunoprecipitation (IP) with antibodies against 5hmC (Active motif rabbit polyclonal against hydroxymethylation cat#39769). For full protocols see17 (link). 10% of the input is also taken prior to immunoprecipitation. In all stages DNA was purified using DNA Clean & Concentrator™ (Zymo Research).The IP and input samples undergo 10 cycles of whole genome amplification following associated protocols (Sigma-Aldrich SeqPlex DNA Amplification Kit). Sequencing libraries are made from 100 ng of DNA for each sample, using the Ion XpressPlus Fragment Library Kit (life Technologies™). The DNA was end repaired, purified and then ligated to Ion-compatible barcoded adapters (Ion Xpress™ Barcode Adapters 1–96: Life Technologies™), followed by nick-repair to complete the linkage between adapters and DNA inserts. The adapter-ligated library was then amplified (10 cycles) and finally size-selected using two rounds of AMPure XP bead capture to size‐select fragments approximately 100–250 bp in length. Samples were then pooled in a 1:1 ratio and sequenced on a single Ion Proton P1 microwell chip (Life Technologies™). Sequence reads were mapped to the reference genome using the supplied TMAP mapping software and the resulting BAM file processed for downstream data analysis.

Harvested pancreas tissues were embedded in OCT. Serial sections were prepared on PEN slides for laser-capture microdissection (LCM). PanIN-2/3 lesions (identified from flanking H&E slides) were laser-captured from serial sections onto Capsure LCM Caps using an ArcturusXT LCM instrument. Caps were extracted using the PicoPure DNA extraction kit from Thermo Fisher Scientific (Waltham, MA). Extracted DNA was amplified using the SeqPlex DNA Amplification Kit (Sigma-Aldrich, St. Louis, MO) and tested for quality using a Bioanalyzer 2100 and run on a 1% gel prior to library preparation. Sample libraries were prepared using the Swift Biosciences Kit (Accel NGS 2 plus) and Agilent Sure Select XT Mouse Exome post-capture kit following manufacturer's instructions. Sample libraries were pooled and run paired-end (2x75 bp) on an Illumina NextSeq 500 for an average of 90M reads per sample.