title: GSM8027908: CHP-134, DMSO, Replicate2, RARA ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: Cross-linking: After desired treatment, cells were incubated with 1% formaldehyde to crosslink the proteins and DNA. Glycine was added to a final concentration of 225 mM to quench the reaction. 20 million cells were used for each sample. Chromatin shearing: The cells were lysed with Covaris Lysis buffer (0.25% Triton X-100, 1 mM EDTA pH 8.0, 140 mM NaCl, 50 mM HEPES pH 7.9, 10% glycerol, 0.5% NP-40). The chromatins were collected by centrifuge and the pellets were washed 2 time with Covaris Wash Buffer (1 mM EDTA pH 8.0, 10 mM Tris HCl pH 8.0, 200 mM NaCl, 0.5 mM EGTA pH 8.0) and 2 times with Covaris Shearing Buffer (0.1% SDS, 1 mM EDTA pH 8.0, 10 mM Tris HCl pH 8.0). Then the chromatins were sheared to 200 – 700 bp in Covaris Shearing Buffer with the ultrasonicator Covaris LE220 (Covaris, Woburn, MA).  Immunoprecipitation: Protein A/G-coated magnetic beads (protein A-coated beads (Diagenode, # C03010020-150) for rabbit antibody; protein G-coated beads (Diagenode, # C03010021-150) for mouse antibody) were washed with 0.5% BSA in PBS (g/ml) and added to the sheared chromatin to preclean the samples by rotation at 4°C for 1 hour. 100 µl of the beads were used for each sample. The magnetic beads were isolated with a magnetic separation rack and the supernatant was collected for immunoprecipitation. Primary antibodies were added to the samples, then the samples were incubated at 4°C overnight with rotation. The antibodies used in this study are HA (Cell Signaling, # 3724; use 40 µl of antibody for each sample in 2 ml of buffer (1:50 dilution)), SMAD4 (R&D Systems, # AF2097; use 20 µg per 20 million cells), and RARA (R&D Systems, # PP-H1920-00; use 20 µg per 20 million cells). On the second day, 100 µl of freshly washed protein A/G-coated magnetic beads were added to each sample. Then the samples were incubated at 4°C for 5 h with rotation. Elution and reverse cross-links: The beads were collected and washed for 1 time with cold Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 150 mM NaCl), 2 times with cold High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 500 mM NaCl), 1 time with cold LiCl Wash Buffer (1 mM EDTA, 10 mM Tris pH 8.0, 250 mM LiCl, 1% NP-40, 1% DOC), and 2 times with cold TE Buffer pH 8.0 (1 mM EDTA, 10 mM Tris pH 8.0). Then the DNA were eluted from the beads with 100 µl of freshly made Elution buffer (0.1 M NaHCO3 and 1% SDS). 4 µl of 5M NaCl and 2 µl of RNaseA (10 mg/mL) (ThermoFisher, # EN0531) were added to each sample and the samples were then incubated at 65°C with shaking overnight. DNA purification: Proteins in the samples were removed by adding 5 µg of proteinase K (ThermoFisher, # AM2548) and shaking at 60°C for 2 hours. Afterward, the DNA was purified with Qiagen MinElute PCR Purification Kit (Qiagen, # 28004). ChIP-seq DNA libraries were prepared using the KAPA HyperPrep Kit (Roche, # KK8504) and sequenced on the Illumina NextSeq 500 system. At least 50 million reads were collected for each sample.