Instrument: Illumina NovaSeq 6000
Strategy: Bisulfite-Seq
Source: GENOMIC
Selection: RANDOM
Layout: PAIRED
Construction protocol: Macro-dissection was performed to decrease the admixture of adjacent normal tissue and to enrich the percentage of tumor material for subsequent RNA extraction. De-paraffinization of FFPE tumor and adjacent normal specimens was performed using QIAGEN de-paraffinization solution (QIAGEN, Valencia, CA). DNA and RNA from 19 tumor and adjacent normal samples was extracted using the AllPrep DNA/RNA FFPE Kit (QIAGEN) following the manufacturer's protocol. In the case of the unavailability of FFPE samples, genomic DNA and RNA were extracted from fresh frozen tumor (n=2) and normal (n=3) samples using the ZR-Duet DNA/RNA MiniPrep extraction kit (ZYMO RESEARCH, Irvine, CA). Quantification was performed with a NanoDrop One™ spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and Qubit™ Fluorometer 2.0 (Qubit, San Francisco, CA) using dsDNA and RNA assay kits. RNA integrity was analyzed using the Tape Station RNA assay kit (Agilent Technologies, Santa Clara, CA). DNA libraries of RRBS were constructed from FFPE tissue samples of 14 tumors/adjacent normal tissue pairs using the Ovation RRBS Methyl-Seq System at The Epigenomics Profiling Core (EpiCore) of MDACC. In preparation, DNA was digested with a restriction enzyme and selected for size based on established protocols used in the EpiCore. Post-adapter ligation ensured enrichment for CpG islands, and DNA was bisulfite-treated, amplified with universal primers, and qualified libraries were then sequenced on Novaseq6000™ and MiSeq sequencers at the UTMDACC ATG