Klenow fragment 3 5 exo minus

Reduced representation bisulfite sequencing (RRBS) was performed as described [62] (link). RRBS reads cover less than 10% of the 28 million CpGs in the human genome [63] (link). Briefly, 1 µg genomic DNA was digested with the methylation insensitive restriction enzyme MspI (NEB). Ends of each restriction fragment were filled in and a 3′ adenosine was added with Klenow Fragment (3′→5′ exo-minus, NEB). Methylated paired-end Illumina adapters were ligated to the ends of the DNA fragments using T4 DNA Ligase (NEB). Fragments between 100 bp and 400 bp were purified by agarose gel extraction. The purified fragments were treated with sodium bisulfite and then amplified by PCR. The final PCR products were sequenced on Illumina HiSeq2000 machines.

The following were added to the bisulfite-treated DNA prepared as above: 5 μl of 10 × NEBuffer 2 (New England Biolabs), 5 μl of 2.5 mM dNTPs (Takara Bio Inc) and 100 pmol of random primer (see Supplementary Table S3); the reaction volume was adjusted to 50 μl with water. The mixture was incubated at 95°C for 5 min, and then cooled and kept at 4°C for 5 min. To the reaction, 1 μl of 50 U/μl Klenow fragment (3′–5′ exo minus) (New England Biolabs) was added and the mixture was incubated at 4°C for 15 min. The reaction temperature was gradually raised to 37°C at a rate of 1°C/min and then maintained at 37°C for 30 min. After heat inactivation of the enzyme at 70°C for 10 min, 50 μl of AMPure XP (Beckman Coulter, Brea, CA, USA) was added, and the reaction mixture was incubated at room temperature for 5 min. The supernatant was removed on a magnetic stand, the beads were rinsed with 75% ethanol and purified DNA was eluted with 13 μl of 10 mM Tris–acetate, pH 8.0. The yield of first-strand synthesis was monitored using Qubit and Qubit dsDNA HS assay kit (Thermo Fisher Scientific), following the manufacturer's instructions.