GSM9570686: ID WT mESC ctcf AID Rep2; Mus musculus; OTHER
Nuclei were extracted using cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 0.5 mM spermidine, 1.5 mM MgCl2, 0.1 mM EDTA, 0.25% IGEPAL CA-630) for 3-6 minutes.  The nuclei were resuspended in 564 µL 1X GpC MTase reaction buffer and equilibrated to 37°C. This was followed by 6 minutes of enzyme treatment (300 µL 1 M sucrose, 34 µL 10X GpC methyltransferase reaction buffer, 3 µL 32 mM S-Adenosylmethionine (SAM), 100 µL GpC methyltransferase (M.CviPI), followed by another 6 minutes of enzyme treatment (50 µL GpC methyltransferase (M.CviPI), 3 µL 32 mM S-Adenosylmethionine (SAM)). An equal volume of pre-warmed 2X stop buffer (20 mM Tris-HCl pH 7.4, 600 mM NaCl, 10 mM EDTA, 1% SDS) was added and mixed well. Proteinase K (200 µg/mL) was added to the GpC methylation reaction and incubated for 4 hours at 56°C, followed by RNase A (100 µg/mL) for 30 minutes at 37°C, followed by an additional round of proteinase K (200 µg/mL) for 30 minutes at 37°C. HMW DNA was extracted using phenol-chloroform and precipitated by isopropanol. Briefly, an equal volume of phenol-chloroform was added to the samples and mixed vigorously. The samples were centrifuged at 13,000 rpm for 10 minutes at room temperature. The aqueous phase was transferred to a clean tube, and 0.7-1 times the volume of isopropanol was added, mixed well, and incubated at room temperature for 30 minutes. The samples were then centrifuged at 13,000 rpm for 10 minutes at 4°C. The DNA pellet was washed with 1 ml pre-chilled 70% ethanol. After removing all the ethanol, the DNA was dissolved in EB buffer and incubated at 37°C for 30 minutes. the DNA fragment was sheared to an average size of 20kb with the Covaris G-Tube (#520079) in an Eppendorf 5415D tabletop microcentrifuge following the manufacture's protocol. Sample size was confirmed on genomic screentape with the Agilent Tapestation system. The sheared DNA (1.5ug) was then input into the SQK-LSK114 library prep from Oxford Nanopore to create the Nanopore library. Components were used from the LSK114 kit along with several buffers and enzymes from New England Biolab (NEB) following the LSK114 protocol from Oxford Nanopore. Following library prep, the sample concentration was verified on the Qubit system using the dsDNA High Sensitivity Kit. The concentration and basepair size were used to calculate Femtomolar concentration for loading. The sample was resuspended with library loading beads and loaded at 150-200 femtomolar concentration onto the Promethion R10.4.1 flow cell according to the LSK protocol. Sample were sequenced on the R10.4.1 Promethion flow cell using the Oxford Nanopore Promethion P24 system with a sequencing run time of 72 hrs. Standard settings were applied during the run setup, special settings were applied and fragments less than 1kb rejected.