title: GSM6500375: 3DRAM-seq mESC rep2; Mus musculus; OTHER
library_construction_protocol: Approximately 2.5x105 fixed mESC were first lysed with 0.2% Igepal-CA630 (Sigma-Aldrich, Cat. N: I3021) for 10min. at RT, washed once with 1x GpC buffer (New England Biolabs, Cat. N.: M0227S) containing 1% BSA (Sigma, Cat. N.: B6917) and subsequently incubated for 3h at 37˚C in a reaction mix containing 60U M.CviPI (New England Biolabs, Cat. N.: M0227S) and 0.6mM SAM (New England Biolabs, Cat. N.: B9003). During the incubation the reaction was substitution with 8U M.CviPI and 1µl of 32mM SAM every hour. Nuclei were washed, permeabilized with 0.5% SDS (Invitrogen, Cat. N:AM9823) followed by a quenching of the SDS with SDS ~1.5% Triton-X-100 (Sigma Aldrich, Cat. N: X100) and a digested with 400U DpnII (New England Biolabs, Cat. N: R0543) overnight at 37˚C. Subsequently, sticky ends were filled by incubating the nuclei for 4h at RT with DNA Polymerase I (New England Biolabs, Cat. N: M0210) and a biotin-14-dATP (Life Technologies, Cat. N: 195245016) containing nucleotide mix in DpnII buffer. Proximity ligation was performed at least for 6h at 16˚C using the T4 DNA Ligase (New England Biolabs, Cat. N.: M0202). Thereafter nuclei and chromatin were digested using 200µg Proteinase K (New England Biolabs, Cat. N: P8107) with 1% SDS followed by reverse crosslinking overnight at 68˚C with 0.5M NaCl, purification by ethanol precipitation and shearing to ~550 bp DNA fragments using a Covaris S220 sonicator. End repaired was performed using the T4 DNA Polymerase (New England Biolabs, Cat. N: M0203) and non-biotinylated nucleotides for 4h at 20˚C. Approximately 0.01% of biotinylated methylation controls are added to the sample and bisulfite conversion was performed using the EZ DNA Methylation-Gold kit (Zymo Research, Cat. N: D5005). For library constructed the Accel-NGS® Methyl-Seq DNA Library kit (Swift Bioscience, Cat. N: 30024) was used according to the manufacturing instructions until the adapter ligation step. After this step, biotin pulldown was performed using MyOne Streptavidin T1 beads (ThermoFisher, Cat. N: 65602) followed by 5 washes with washing buffer containing 0.05% Tween-20 (Sigma-Aldrich, Cat. N: P9416) and two additional washes with low-TE water. Final library amplification was performed in 5 separate reactions using the EpiMark Hot Start Taq (New England Biolabs, Cat. N: M0490) with Methyl-Seq Indexing Primers (Swift Bioscience, Cat. N: 36024) with the following PCR program: 95˚C 30s; {95˚C 15s, 61˚C 30s, 68˚C 80s} x10-11; 68˚C 5min; Hold at 10˚C. The different reactions were pooled, Streptavidin T1 beads were pelleted on a magnetic rack and the prepared libraries within the supernatant were purified using 0.65x AMPure XP beads (Agencourt, Cat. N: A63881) to reach an average fragment size of approximately 500bp.