Epimark bisulfite conversion kit

Cells were harvested 48 h after siRNA transfection, and genomic DNA was extracted using phenol-chloroform. Genomic DNA (2 µg) was modified with bisulfite using the EpiMark® Bisulfite Conversion Kit (New England Biolabs) according to the supplier’s protocol. Bisulfite-modified DNA (40 ng) was amplified by PCR using Platinum™ Taq DNA polymerase and primer pairs (see below) to cover the BRCA1 promoter (GenBank Accession No. U37574). The PCR products were run on a 1% agarose gel, excised, extracted using QIAquick Gel Extraction Kit (Qiagen), and subcloned into pCR™ 2.1-TOPO® TA vector using TOPO® TA Cloning® Kit (ThermoFisher Scientific). At least 10 clones of each PCR product were subjected to Sanger sequencing, and CpG methylation was analyzed by Quantification Tool for Methylation Analysis (QUMA)^{48 (link)}. PCR primers specific for bisulfite-converted BRCA1 promoter were designed using MethPrimer software^{49 (link)}. The positions of the primers are shown in Fig. 3a.
Promoter region 1422–1967:
Forward: 5′-AGATTGGGTGGTTAATTTAGAGTTT-3′
Reverse: 5′-ATAATATCCCCCTCAAAACATATTC-3′
The reverse primer was used for Sanger sequencing.

500 ng of genomic DNA (from recombinant CHO and HeLa cell lines) was treated with sodium bisulfite according to Epimark bisulfite conversion kit (New England Biolabs). Fragments adjacent to the 5′ and the 3′ recombinant HsMar1 TIRs were amplified using 50 ng of treated DNA and the m-primers designed by Methprimer (Additional file 1: Table S1). PCR products were cloned in pGEMT and sequenced by Eurofins. Analysis of CpG methylations was done using the QUMA software. ENCODE RRBS analysis were used to detect methylation environment (ENCFF001TMU & ENCFF001TMV) [40 (link)].

For D. pigrum CDC4709-98 (aka KPL1934), the presence of 5-methylcytosine in the predicted methylation motif GCNGC was assessed as previously described (127 (link)). Briefly, gDNA harvested with a Masterpure Complete DNA/RNA purification kit was bisulfite treated using an EpiMark bisulfite conversion kit (NEB, Ipswich, MA)—both according to manufacturer’s instructions, except for a final elution volume of 20 μl in the EpiMark kit. We then selected two genomic regions: each ≤700 bp containing ≥4 GCNGC motifs. We PCR amplified each region from 1 μl of the converted gDNA using TaKaRa EpiTaq HS for bisulfite-treated DNA (TaKaRa Bio USA, Mountain View, CA) according to the manufacturer’s instructions with the primers designed by MethPrimer: oKL732 (5′-AAGTTTATTTTTTTGAGTTTGTTG-3′), oKL733 (5′-TACCCATAAAATTATCACCTTC-3′), oKL734 (5′-ATTGATTTAGTAATTTTTTTGGAATAT-3′), and oKL735 (5′-TAAATAACTCTACAAAAAACTCAACTTACC-3′). After amplicon purification with a QIAquick PCR purification kit (final elution, 40 μl; Qiagen; Germantown, MD), we used Sanger sequencing (Macrogen, USA) of each PCR product to detect cytosine methylation within the predicted motif. Additional m5C-based modified motif analysis was carried out for Dolosigranulum pigrum KPL3250 using MFRE-Seq, as previously described (128 (link)).

The methylation status of the core region of the LHCB1.2 promoter was semi-quantified by combined bisulfite restriction analysis (COBRA) adopted from^{54 (link)}. Approximately 1 μg of genomic DNA was treated with the EpiMark Bisulfite Conversion Kit (NEB, E3318), and the LHCB1.2 core promoter region was amplified from the bisulfite-converted DNA using the primer pair pLHCB1.2-F (− 302 bp) and pLHCB1.2-R (13 bp) (see Supplementary Table S3 online). The resulting PCR product was gel-purified and digested with restriction enzymes as indicated. Then the digested PCR product was separated on an 3% agarose gel and the DNA band was visualized by ethidium bromide staining.