Truseq methyl capture epic library preparation kit

As input, 500 ng of total DNA per sample were utilized for preparing targeted methylseq libraries with Illumina TruSeq Methyl Capture EPIC Library Preparation Kit (Illumina (San Diego, CA, USA)). All steps were performed as recommended in Illumina user document 1000000001643 v01 May 2017. Furthermore, one additional purification step was introduced at the end of the standard procedure, using 1x Agencourt® AMPure® XP Beads (#A63881; Beckman Coulter, Inc.). The KAPA Hifi HotStart Uracil+Ready Mix 2x enzyme is needed to amplify the enriched libraries but is not substitutable and not included with the kit. DNA libraries were indexed and amplified with 11–13 cycles of PCR. Fragment length distribution of individual libraries was monitored using Bioanalyzer High Sensitivity DNA Assay (5067-4626; Agilent Technologies (Santa Clara, CA, USA). Quantification of libraries was performed by use of the Qubit^® dsDNA HS Assay Kit (Q32854; ThermoFisher Scientific).

DNA samples (10 ng/µl, 500 ng total) were sheared using the S220 Focused-ultrasonicator (Covaris) to generate dsDNA fragments. The D1000 ScreenTape System (Agilent) was used to ensure >60% of DNA fragments were between 100 and 300 bp long, with a mean fragment size of 180–200 bp. The methylation analysis was performed using the TruSeq Methyl Capture EPIC Library Preparation Kit (Illumina) using the manufacturer’s protocol. Twelve samples were pooled for sequencing on the HiSeq4000 Illumina Sequencing platform (single end 150 bp read) using two lanes per library pool. Technical replicates were performed for cell line data to assess assay reproducibility (R² = 0.97). The reads were trimmed (TrimGalore v0.4.4), aligned to the bisulfite converted human reference genome (GRCh38/hg38) and methylation calling was performed using Bismark (v0.22.1) using standard parameters. Quality control (QC) reports were compiled using FastQC (0.11.4) and MultiQC (v1.7). The position of the CpG island (hg38-chr9:130444478–130445423) overlapping with the TSS of ASS1 (GRch38 chr9:130444200–13044780) was obtained from Ensembl.