endothelial cells from umbilical cords newborn healthy humans
TFEB (#4240, CST)
Sequenced DNA Library
For ChIP-seq, approximately 2 x 10E7 cells were cross-linked by addition of formaldehyde to 1% for 10 min at RT, quenched with 0.125 M glycine for 5 min at RT, and then washed twice in cold PBS. The cells were resuspended in Lysis Buffer 1 (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100 and protease inhibitor) to disrupt the cell membrane and in Lysis Buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and protease inhibitor) to isolate nuclei. The isolated nuclei were then resuspended in SDS ChIP Buffer (20 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS and protease inhibitors). Extracts were sonicated using the Bioruptor H Twin (Diagenode) for 2 runs of 10 cycles [30 sec ‘‘ON’’, 30 sec ‘‘OFF’’] at high power setting. Cell lysate was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was diluted with ChIP Dilution Buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton) before immunoprecipitation step. Magnetic beads (Dynabeads® Rat Anti-Mouse IgM for anti-PolII-phospho-S5, Dynabeads®Protein G for all the other ChIPs, Life Technologies) were saturated with PBS/1% BSA and the samples were incubated with 2 micrograms of antibody overnight at 4°C on a rotator. Next day samples were incubated with saturated beads for two hours at 4°C on a rotator. Successively immunoprecipitated complexes were washed five times with RIPA buffer (50 mM Hepes-KOH pH7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0,7% Na-Deoxycholate) at 4°C for 5 minutes each on a rotator. Elution Buffer was added and incubated at 65°C for 15 minutes. The decrosslinking was performed at 65°C overnight. Decrosslinked DNA was purified using QIAQuick PCR Purification Kit (QIAGEN) according to the manufacture’s instruction. For ChIP-seq, approximately 10 ng of purified ChIP DNA were end-repaired, dA-tailed, and adapter-ligated using the NEBNext ChIP-Seq Library Prep Master Mix Set (NEB), following manufacturer instructions.