NextSeq 500 paired end sequencing; Bait-capture based single molecule footprinting of different mouse cell lines
Bait-capture based single molecule footprinting of different mouse cell lines
The myoblast (C2C12) cells were grown on collagen coated plates in low glucose DMEM medium/F10 nutrient mix supplemented with 20% FBS and 2 mM L-glutamine. Medium was changed daily and cells were split every second day. As described in Kleinendorst and Bazaghi et al., 2021 (https://doi.org/10.1038/s41596-021-00630-1). In short, cells were harvested using trypsin and washed twice with ice cold PBS. For one reaction 250,000 cells were resuspended in 1 mL ice-cold hypertonic lysis buffer (10 mM Tris (pH = 7.4), 10 mM NaCl, �3mM Mgcl2, 0.1 mM EDTA, 0.5% NP40), incubated on ice for 10 min and spun at 1,000x g at 4�C for 5 min. Nuclei were washed in the same buffer without NP40 and spun again. Nuclei were resuspended in 1x M.GpC buffer (NEB, #M0227L). For the GpC methyltransferase treatment, freshly made GpC methyltransferase mix (1x M.GpC buffer, 300 mM sucrose, 64 _M SAM) and 200U M.CviPI (NEB, #M0227L) were added and incubated at 37�C for 7.5 min. The reaction was supplemented with 100U of M.CviPI and 128 pmol of SAM for a second incubation round at 37�C for 7.5 min. For DNMT TKO samples, where dual enzyme footprinting was applied, another round of footprinting was done by adding 10 mM MgCl2, 128 pmol of SAM and 60U of M.SssI (NEB, #M0226L) and incubation at 37�C for 7.5 min. After fooprinting, prewarmed stop solution (20 mM Tris, 600 mM NaCl, 1% SDS, 10 mM EDTA) and proteinase K were added and incubated overnight at 55�C. The next day, DNA was extracted using phenol-chloroform and treated with RNase A at 37�C for 30 min. As described in Kleinendorst and Barzaghi et al., 2021 (https://doi.org/10.1038/s41596-021-00630-1). In short, for making the Whole Genome Bisulfite Sequencing library with targeted enrichment, DNA was fragmented into 300 bp fragments via sonication using Covaris model S2. For library preparation and targeted enrichment of cis-regulatory elements, the SureSelect XT Mouse Methyl-Seq Kit Enrichment System for Illumina Multiplexed Sequencing Library protocol (Agilent Technologies, version E0, April 2018) was used as described in Kleinendorst and Barzaghi et al., 2021. Bisulfite conversion was performed using the ZYMO EZ DNA Methylation-Gold Kit (Zymo, #D5005) according to the manufacturers protocol. The bisulfite-converted library was PCR amplified and indexed using the SureSelect XT Mouse Methyl-Seq Kit (Agilent, # G9651A).