GSM6172820: DKD, rep4, MBL2; Homo sapiens; OTHER
UMI-4C was performed essentially following a described protocol (Schwartzman et al., 2016). More specifically, 2.5 million HepG2 cells were seeded in 6-well plates. The day after, the cells were fixed by addition of PBS 1% FA and incubation for 10 min at RT. The reaction was stopped by adding glycine to a final concentration of 0.125M and incubation for 5 min on ice. The cells were then collected by scraping, washed with PBS and resuspended in 500 µL of lysis buffer (10 mM TRIS pH 8, 10 mM NaCl, 0.2% NP-40) and incubated for 30 min on ice. Nuclei were isolated by centrifugation for 5 min at 760 g and resuspended in 500 µL of DpnII buffer (NEB). 15 µL of SDS 10% were added to the tube and incubated for 60min at 37ºC shaking at 900rpm. Then, 150 µL of 10% Triton X-100 were added and the sample was incubated for 60 min at 37ºC. Afterwards, digestion of the nuclei was achieved by addition of 400U DpnII (NEB) and o/n incubation at 37ºC. The next morning, 200U DpnII were added again and the sample was incubated for 4 more hours at 37ºC, afterwards DpnII was inactivated for 20min at 65ºC. Digested nuclei were pelleted by centrifugation for 5 min at 600 g and resuspended in 5.7mL milli-Q H2O. Then, 700 µL T4 ligation buffer (Promega) and 20 µL T4 ligase (Promega, 3 U/µL) were added. The samples were incubated overnight at 16ºC. The samples were then digested with 5 µL RNAse A (10 mg/µL) and 10 µL proteinase K (10 mg/mL) for 30min at 37ºC and decrosslinked o/n at 65ºC. DNA was purified by phenol-chloroform extraction and ethanol precipitation and quantified with the Qubit™ dsDNA HS Assay Kit (Thermo Fisher). A total of 5 µg of DNA were sonicated with a Covaris S2 instrument with the following parameters: duty cycle 10%, intensity 4, cycles per burst 200 and time 60 s. Sequencing libraries were prepared as described (Schwartzman et al., 2016). The procedure is based in a nested PCR using oligonucleotides aligned to a specific region functioning as “bait” or “point of view” for capturing contacts.