Epixplore methylated dna enrichment kit

Genomic DNA was isolated from cells using the AllPrep RNA/DNA Mini Kit (Qiagen, Cat# 80204) according to manufacturer’s instructions. DNA was quantified by a NanoDrop 1000 spectrophotometer and 1 μg DNA was sheared by sonication to an average size of around 200 bp. Methylated DNA was enriched from sheared genomic DNA using the EpiXplore Methylated DNA Enrichment Kit (Takara Bio, Cat# 631962) according to manufacturer’s instructions. Enrichment was measured by qRT-PCR using primers designed to various regions of HLA-A, HLA-B, and HLA-C (Integrated DNA Technologies). Primers are listed in Supplementary Data S5.

For the preparation of CpG‐methylated nucleosomal DNA, 50 μg·mL⁻¹ of the CpG146 DNA was incubated at 37 °C for 16 h with M.SssI (6 units·μg⁻¹ DNA), in 10 mm Tris‐HCl buffer (pH 8.0), containing 50 mm NaCl, 2.5 mm EDTA, and 640 μm SAM. To separate the reacted nucleosomal DNA from the M.SssI, the reaction was extracted with phenol–chloroform–isoamyl alcohol (25 : 24 : 1), and the DNA was ethanol‐precipitated. A small portion of the reacted DNA was digested with Eco72I (Thermo Fisher Scientific; cat. ER0361) or MspJI (NEB, cat. R0661S), at 1.8 units·pmol⁻¹ DNA and 0.45 units·pmol⁻¹ DNA, respectively. The CpG‐methylated CpG146 DNA was also used in the binding assay with MBD2, using an EpiXplore methylated DNA enrichment kit (Takara Bio, Shiga, Japan; cat. 631963). In each MBD2‐binding reaction, the histidine‐tagged MBD2 protein (5 μg) from the kit, which was immobilized onto 4 μL of TALON resin, was incubated with 500 ng of the 146‐bp nucleosomal DNA at 25 °C for 1 h. The incubated samples were washed four times, using the 1× binding/washing buffer of the kit, and the nucleosomal DNAs were eluted with elution buffer (high). The nucleosome DNA of each fraction was ethanol‐precipitated and suspended in 20 μL of 10 mm Tris‐HCl buffer (pH 8.0) containing 100 μm EDTA. One quarter (5 μL) of each fraction was loaded onto a 5% polyacrylamide gel and analyzed by electrophoresis.

RPA was performed using a TwistAmp™ Basic kit (TwistDx, Maidenhead, UK) as follows: a freeze-dried component was reconstituted with 29.5 µl of rehydration buffer and 2.5 µl of each primer (10 µM). A 13.6 µl aliquot was mixed with DNA (20 ng unless stated) to create a pre-reaction mixture. The required blocking agent (0.25–4 µM ORN, 1 µl of CRISPRi RNP complex, 20 or 40 ng of LexA, 0.25–2 µg of MBD2 from the EpiXplore™ Methylated DNA Enrichment Kit (Takara Bio, Shiga, Japan), or 0.5–2 µM 2’3’ddC-modified ODN) and nuclease-free water were added to the pre-reaction mixture to a final volume of 19 µl and incubated at 37 °C for 5 min (pre-incubation). Thereafter, 1 µl of MgOAc (280 mM) was added and the reaction was incubated at 37 °C for a further 30 min. RPA products were purified using a PCR/Gel DNA purification kit (Nippon Genetics, Tokyo, Japan), electrophoresed on 2% or 3% agarose gels, and sequenced if required. DNA gel images were acquired using AE-6905H Image Saver HR (ATTO, Tokyo, Japan) and the DigiDoc-It system (UVP, Cambridge, UK). DNA sequencing data were analyzed using Applied Biosystems Sequence Scanner Software v2.0 (ThermoFisher Scientific, Waltham, MA, USA).