Kapa hifi uracil

CMS-IP was performed similar to pervious described (Huang et al., 2012 (link); Pastor et al., 2011 (link)). Briefly, genomic DNA from in vitro cultured pro-B cells was isolated with PureLink Genomic DNA kit (Thermo Fisher) and were spiked-in with cl857 Sam7 λDNA (Promega,Madison, WI) and PCR-generated, hmC-containing puromycin-resistant gene at a ratio of 200:1 and 100,000:1, respectively. DNA was sheared with a Covaris E220 (Covaris), end-repaired, A-tailed, ligated with methylated Illumina adaptors (NEB, Ipswich, MA), and bisulfite-converted (MethylCode Bisulfite Conversion Kit, Thermo Fisher). Bisulfite-converted DNA was denatured and immunoprecipitated with anti-CMS serum. Immunoprecipitated DNA was PCR-amplified with barcoded primers (NEBNext Multiplex Oligos for Illumina, NEB) for 15 cycles with Kapa HiFi Uracil+ (Kapa Biosystems, Wilmington, MA). Resulting libraries were sequenced with a HiSeq2500 system for 50 bp paired-end reads (Illuminia, San Diego, CA). The sequence reads were mapped to mm9 with Bismap, and CMS-enriched genomic regions were identified using the ‘findPeaks' command in HOMER with the 'histone’ mode and default parameters (Heinz et al., 2010 (link)).

Genomic DNA from each tissue was isolated using the NucleoSpin^® Tissue kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. DNA concentration was estimated by PicoGreen^® (Thermo Fisher Scientific, Waltham, MA, United States). Libraries were generated using the TruSeq^® DNA PCR-Free Library Preparation Kit (Illumina) including a step of bisulfite treatment. After ligation of adapters, samples were converted with EpiTect Bisulfite Kits (Qiagen) and finally PCR amplified with KAPA HiFi Uracil+ (Kapa Biosystems). WGBS was performed on an Illumina Hiseq 2000 (San Diego, CA, United States) to generate 100-base paired-end reads.

In total, 5 regions were selected for validation, which covered 18 CpGs interrogated by the 450 k array. Primers were designed for bisulfite-converted DNA using MethPrimer [37 ]. In total, 10 ng of bisulfite-converted FFPE extracted DNA from four trios was used to amplify the specific genomic regions. The hot-start enzyme KAPA HiFi Uracil + (KAPA Biosystems Inc, Wilmington, MA, USA) was used for PCR and products were cleaned using magnetic beads (Beckman Coulter Inc, Brea, CA, USA) and quantified using Picogreen reagents. Samples were tagged and pooled prior to sequencing on the Illumina MiSeq according to the manufacturer’s instructions.
Raw MiSeq paired-end reads were mapped to human genome build hg19 with Bismark v0.9.0 [38 ] using Bowtie 2 [39 ] as the aligner. Methylated and unmethylated base counts were generated with the bismark_methylation_extractor utility and exported as BedGraph files for further analysis and display in Integrative Genomics Viewer [40 ]. Aligned BAM files were sorted and indexed with SAMtools [41 ] for assessment of the regions of interest in Integrative Genomics Viewer. The number of C reads (methylated prior to conversion) was divided by the total number of reads per bisulfite-sequenced CpG site to discern the percentage methylation. These were then compared with the respective 450 k β-values to compare platforms.