title: GSM3864901: D_2_combined; Mus musculus; OTHER
library_construction_protocol: About 1,000 cells were transferred into a 0.2 μL PCR tube containing 7 μL of ice-cold lysate buffer (50 mM Tris-HCl (pH7.4), 50 mM NaCl, 0.25 mM EDTA, 10 mM DTT, 0.25 mM PMSF and 0.5% NP-40, plus 2 pg λDNA). After gently vortex, the cell lysate was kept on ice for 10 min. The GpC methyltransferase M. CviPI and SAM were then added to the lysate to a final volume of 10 μL containing 1 U/μL M. CviPI and 160 μM SAM. The in vitro methylation of nuclei was performed by incubating the mixture in a thermocycler at 37℃ for 45 min followed by heating at 65℃ for 25 min to inactive the enzyme activity. After in vitro methylation, 1 μL of 20 mg/mL protease (Qiagen) was added and the mixture was incubated for 3 hrs at 50℃ to release genomic DNA. The released genomic DNA was then bisulfite converted using the EZ-96 DNA Methylation-Direct MagPrep (Zymo) according the manufacturer's instructions. Afterwards, the purified DNAs were annealed using random nonamer primers with a 5'-biotin tag (5′-Biotin-CTACACGACGCTCTTCCGATCTNNNNNNNNN-3′) in the presence of Klenow fragments (3'-5' exo-, NEB). Then, the primers were digested by exonuclease I (NEB) and the DNA was purified using Agencourt Ampure XP beads (Beckman Coulter). Dynabeads M-280 Streptavidin (Invitrogen) were then used to immobilize the newly synthesized biotin-tagged DNA strands, and the original bisulfite-converted DNA templates were removed. Second DNA strands were synthesized using Klenow fragment with random nonamer primers (5′-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3′). After washing, the beads were used to amplify libraries using 15 cycles of PCR with the universal primer and index primer (NEB). The amplified libraries were purified with Agencourt Ampure XP beads twice. Fragment from 300 bp to 800 bp were selected by argarose gel electrophoresis and purified by Zymoclean Gel DNA Recovery Kit (Zymo). Finally, libraries were pooled and sequenced on the Illumina HiSeq 2500 sequencer for 150-bp paired-end sequencing.