Epitect bisulfite conversion kit

For methylation analyses, DNA was purified from untreated or 5-aza-dCt treated cells or thyroid samples as previously reported [19 (link)] and quantitated by absorbance at 260 nm. To deaminate unmethylated cytosines, and thus distinguish methylated from unmethylated alleles, DNA (1 μg) was converted using EpiTect Bisulfite conversion kit (QIAgen, Hilden, Germany) according to the manufacturer's instructions. Bisulfite-converted DNA (10–30 ng) was used as template in a 25-μL PCR reactions containing 10 mM dNTPs, 50 mM MgCl_2, 20 mM Tris-HCl, 50 mM KCl, 1,5U Platinum Taq DNA polymerase (Life Technologies) and 10 pmol of specific primers. Primers flanking the CpG sites located within intron1 (designed IR) or within the promoter region of ABI3 (designed R1, R2 and R3) were designed (Figure 1B). Primers sequence and PCR conditions are described in Supplementary Table S1.
PCR products were then cloned into pCR2.1-TOPO vector using TOPO TA Cloning kit (Life Technologies) according to the manufacturer's instructions. Nearly 8–12 independent colonies were picked randomly and the inserts were amplified and sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). After bisulfite treatment, the degree of methylation at each CpG site in the individual regions were determined from the ratio of methylated cytosines to total cytosines and expressed as percentage.

Prior to library preparation, unmethylated lambda phage DNA and SssI‐methylated T7 DNA were spiked in to genomic DNA samples to control bisulfite conversion rates. For RRBS, 20 μg of DNA was digested for 18 h by MspI and separated on 2% agarose gels. Digested DNA was excised at 150–250 and 250–350 bp. Digested DNA samples were end‐repaired and A‐tailed using the NEB Ultra Illumina library preparation kit (E7370). DNA fragments were ligated to methylated adapters (NEB E7535) following the NEB Ultra protocol. After adapter removal using Ampure XP beads (Beckman Coulter), DNA was converted using the Qiagen Epitect bisulfite conversion kit following the FFT sample protocol. After conversion, library PCR amplification was performed with 10 cycles following NEB Ultra kit protocol and cleaned up using Ampure XP beads. Two libraries were generated each for 150‐ to 250‐bp and 250‐ to 350‐bp inserts.

Genomic DNA was isolated from TGS‐01 and TGS‐04 cells, and bisulfite treatment was performed using an EpiTect Bisulfite Conversion Kit (Qiagen). Using the bisulfite‐treated genomic DNA as a template, PCR was carried out using EpiTaq HS (Takara Bio). The PCR products were analyzed by sequencing or agarose gel electrophoresis. The primers used for the PCR are listed in Tables S6 and S7.

WGBS of Atf7ip and Zmym2 KO mESCs was performed as described previously^{64 (link)}. In short, 10 µg genomic DNA was sonicated to a length of approximately 400–500 bp. For each sample, 2 µg sheared genomic DNA was mixed with 10 ng equimolar pooled sonicated methylated phage T7 and unmethylated phage Lambda DNA. Adapter-ligation was carried out with the NEBNext Ultra II kit (NEB E7645L) using methylated adaptors (NEB, E7535S), before bisulfite conversion using the Qiagen Epitect bisulfite conversion kit, according to the manufacturer’s instructions. After conversion, libraries were amplified for 10 cycles using the Pfu TurboCx Hotstart DNA polymerase (Agilent) and the NEB dual index primers (NEB, E7600S). PCR reactions were run with the following parameters: 95 °C for 2 min, 98 °C for 30 s, followed by 10 cycles of 98 °C for 15 s, 65 °C for 30 s and 72 °C for 3 min, ending with 5 min at 72 °C. The PCR reactions were cleaned up using 1.2× AMPure XP beads (Beckman Coulter) and eluted in 20 µl EB buffer (Qiagen). Library quality was checked on an Agilent TapeStation and sequencing was done on an Illumina NovaSeq 6000 machine.

First, DNA was isolated from 3–5 mL of peripheral blood samples employing the kit QIAamp^® DSP DNABlood Mini (QIAGEN, Hilden, Germany); the evaluation of purity and concentration of the isolated DNA was determined with a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA was stored at −20 °C. A total of 1000 ng of DNA was utilized in the bisulfite conversion treatment using the Epitect Bisulfite Conversion Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. In the final process, each sample of bisulfite-converted DNA was suspended in 60 µL of elution buffer for the pyrosequencing technique.

Genomic DNA (gDNA) was extracted from MUCs from the control and treatment groups using a Flexi DNA Kit (Qiagen), followed by a bisulfite conversion reaction using an EpiTect Bisulfite Conversion Kit (Qiagen) according to the manufacturer's protocol. Nested-MSP was used to detect the methylation pattern of studied genes with a Qiagen MSP Kit (n = 3). The primers targeting the CpG islands of promoter regions of studied genes for nested-MSP were included in Table 1. The products of nested-MSP were analyzed by electrophoresis and ChemiDoc-It® 2 imaging system. To compare the methylation level of studied genes, expression of unmethylated studied gene was normalized in the control and treated MUCs.