GSM8386429: HEK293T_ChIPseq_ZNF143_Proteintech_dTAG_rep2; Homo sapiens; ChIP-Seq
Formaldehyde (final concentration 1%, Electron Microscopy Sciences) was added directly to the plates containing 10 mL of media and incubated for 10 minutes at 37°C in the incubator to fix the cells. The reaction was then quenched by adding 0.125M glycine (final concentration) to the plates, swirling, and incubating for an additional 10 minutes at 37°C. The plates were washed once with 10 mL and then with 5 mL of ice‐cold PBS containing protease inhibitors (cOmplete Roche, Sigma‐Aldrich). Subsequently, the cells were scraped into 5 mL of ice‐cold PBS containing protease inhibitors and transferred to a 15 mL tube. The process was repeated to collect all the cells, and the pellet was transferred to a 1.5 mL tube and stored at -80°C after snap freezing in liquid nitrogen. For ChIP, the frozen pellets were thawed on ice for 10 minutes. The cells were then lysed by resuspending in 1 mL of cold sonication buffer per 1 x 108 cells (0.5% SDS, 0.01M EDTA, 50mM Tris-HCl pH 8.0, and 1X protease inhibitors). The lysate was divided to obtain approximately 200 to 300 μL per tube and sonicated for 15 sec ON, 15 sec OFF, for 30 minutes at 25% amplitude using a Qsonica sonicator. The samples were then spun at ~17000x g in a mini centrifuge for 10 minutes at 4°C, and the supernatant was collected. The supernatant was diluted (at least 5 times) to achieve a concentration of one million cells per 200 μL lysate to reduce SDS concentration with ChIP dilution buffer containing protease inhibitors (0.01% SDS, 1.2mM EDTA, 16.68 mM Tris–HCl pH 8.0, 167mM NaCl, 1.1% Triton X-100, and 1X protease inhibitors). Four million cell equivalents of cell lysate were aliquoted, and appropriate amounts of antibody were added (4 μL of anti‐CTCF antibody (3418S, Cell Signalling, 91ng/μl) or 18μL of anti-ZNF143 antibody (16618-1-AP, Proteintech, 500 ng/µl)). The samples were incubated overnight with rotation at 4°C. The immune complex was collected by incubating with 40 μL Protein A + Protein G beads (Dynabeads Protein A and Protein G, Invitrogen) on a rotisserie at 4°C for 90 minutes. The beads were washed once each with low salt immune complex buffer (0.1% SDS, 1% Triton x-100, 2mM EDTA, 150mM NaCl, 20mM Tris HCl pH 8.0), high salt immune complex buffer (0.1% SDS, 1% Triton x-100, 2mM EDTA, 500mM NaCl, 20mM Tris-HCl pH8.0), LiCl immune complex buffer (0.25M LiCl, 1% NP-40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl pH8.0), and then with 1x TE (10mM Tris-HCl, 1mM EDTA pH8.0), all containing 1x protease inhibitors. To elute the immunoprecipitated complexes, 150 μL of elution buffer (10mM Tris-HCl pH 8.0, 300mM NaCl, 55mM EDTA, 0.5% SDS) was added, followed by 1 μL RNAse A, and incubated at 37°C for 10 minutes. This was followed by the addition of 5 μL Proteinase K and overnight incubation at 65°C. The DNA was purified after removing the beads with a MinElute PCR purification kit (Qiagen), and eluted DNA in a 52 μL elution buffer provided in the kit. ChIP‐seq libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB) and NEBNext Multiplex Oligos for Illumina (NEB) index primers, following the manufacturer's protocols. Libraries were assessed on a TapeStation and Qubit and sequenced on Illumina NextSeq 500 generating paired-end reads.