Amplicon based single molecule footprinting in TC-1 DNMT TKO mESCs after RMCE and after 24h p300 inhibition
Amplicon based single molecule footprinting in TC-1 DNMT TKO mESCs after RMCE and after 24h p300 inhibition
Mouse ES cells (129 WT, DNMT TKO, TET TKO and F1 hybrid cells (129/CAST)) were cultured on 0.2% gelatin-coated plates in ES medium (DMEM, supplemented with 15% Fetal Bovine Serum (FBS), LIF, 2-Mercaptoethanol, 2 mM L-Glutamine and 1x non-essential amino acids) at 37°C and 5% CO2. Medium was changed daily and cells were split every second day. We use previously described TC-1 ES cells that have a landing pad for targeted insertion using Recombination Mediated Cassette Exchange (RMCE). For targeted insertion of the DNA libraries into the mouse genome RMCE was used, as previously described [Krebs et al., 2014]. Shortly, TC-1 DNMT TKO ES cells were selected under hygromycin (250 μg/ml, Roche, Switzerland) for 14 days. After, 50M cells were nucleofected (Amaxa 4D-Nucleofector Core Unit, Lonza) with 300 µg DNA libraries containing plasmid and 180 µg pIC-CRE plasmid. After 2 days, cells were selected with 3 µM ganciclovir (Selleckchem, Catalog No. S1878) for 10 days. Single Molecule Footprinting (SMF) was performed as previously described [Kleinendorst et al., 2021a; Sönmezer et al., 2021]. In short, cultured cells were harvested using trypsin and washed twice with 1x PBS. Cells were counted and 250,000 cells were used per reaction. Cell pellets were resuspended in ice-cold lysis buffer, incubated on ice for 10 min and spun at 1,000x g at 4°C for 5 min. 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 μM SAM (NEB, #B9003S)) and M.CviPI (NEB, #M0227L) were added and incubated at 37°C for 7.5 min. Prewarmed stop solution 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.  For making the library for targeted SMF at the ectopic site, the extracted DNA was bisulfite converted using the Qiagen Epitect bisulfite kit as described in [Kleinendorst et al., 2021a]. Bisulfite converted DNA was amplified using KAPA HiFi Uracil+ (Roche) ([95 °C, 4 min] × 1; [98 °C, 20 s; 60 °C, 15 s; 72 °C, 20 s] × 35, [75 °C, 5 min] × 1; 4 °C, hold) and purified using AMPure XP beads (0,75x, Beckman Coulter). The sequencing library was prepared using the NEBNext DNA Ultra II Library Prep Kit.