GSM9241586: HCT116 cells, 0hrs, Pore-C rep1 and rep2; Homo sapiens; OTHER
Protocol was adapted from the published method (Xiao et al. 2022) with additional RNA digestion using 10U of RNase A/T1 Mix (ThermoScientific, cat. no. EN0551) at 37°C for 20min before Pronase decrosslinking. After decrosslinking the DNA was extracted by Phenol/Chloroform extraction and ethanol precipitation as described before. 4.5x106 endogenous Halo-SON SRRM2-SNAP-FKBP HCT116 cells were incubated with 1µM HaloPROTAC3 (MedChemExpress, cat #HY-111997) and 1µM dTAG13 (Tocris, cat. no 6605) for 0 hours (untreated) 6 hours, 12 hours or 18 hours as well as DMSO for 18 hours. After the incubation the cells were fixed with 4mL 1% formaldehyde (final) in PBS (Pierce™ 16% Formaldehyde (w/v), Methanol-free, cat. no. 28908) for 10 min at room temperature and blocked with 125mM Glycine. Pellet of the fixed cells was frozen at -80°C for at least 1 hour. Nuclei were then isolated at 4°C for 30 min with Cytoplasm lysis buffer (10mM Tris-HCl, 10mM NaCl, 0.2% Igepal, 1x cOmplete protease inhibitors (MERCK, cat. no. COEDTAF-RO)). Cells were then permeabilized with 0.5% SDS at 62°C, which was subsequently inactivated 2% Triton-X-100 (37°C, 15min). DNA was then fragmented in-situ using 100U of NlaIII (NEB, cat. no. R0125S) in 1x CutSmart buffer (NEB, cat. no. B6004S) at 37°C for 16 hours. Next, The restriction enzyme was heat inactivated at 62°C for 20 min and restriction fragments ligated for 4 hours in 16°C followed by 1 hour in 25°C using 4000U of T4 ligase (NEB, cat. no.µg M0202L) in 1x T4 ligase buffer (NEB, cat. no. B0202S) with 1.25% Triton-X-100 and 10µg of BSA. The sample was decrosslinked for 16 hours at 63°C by addition of SDS to 0.5% and proteinase K to 1mg/mL (Invitrogen, cat. no. 25530-049). DNA was then extracted using phenol:chloroform: isoamyl alcohol (25:24:1). RNA in purified sample was digested with 10U of RNase A/T1 Mix (ThermoScientific, cat. no. EN0551) at 37°C for 20min followed by inactivation and second round of decrosslinking for 40min at 37°C with 2mg/mL pronase in 1x pronase buffer (5mM Tris, 10mM EDTA, 0.5% SDS). ONT native barcoding library preparation according to ONT protocol SQK-NBD114.24