GSM8385517: Si1235,H2AZ,ChIP,rep2; Homo sapiens; ChIP-Seq
HCC1954 cells were transfected with ASOs as indicated. After 72h, cells were treated with 1% formaldehyde for 10 min at RT prior to the addition of glycine at the final concentration of 0.125 M for 5 min. Cells were washed twice with PBS and treated with Buffer A (10 mM Tris-HCl 7.5, 10 mM NaCl, 0.5% NP-40) with 1X Protease Inhibitor Cocktail (PIC) for 10 min on ice. Washed once with Buffer A+PIC, Cells were resuspended in nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS). The nuclei were sonicated for several rounds until the de-crosslinked DNA fragment was about 200-500 bp with Bioruptor Pico (5 cycles in our case). Centrifuge 12000 g at 10°C for 15min. For 100 μl cleared sonicated chromatin, add 900 μl ChIP dilution buffer (0.01% SDS, 1% Triton-X100, 1 mM EDTA pH 8.0, 16.7mM Tris-HCl pH 8.0, 167 mM NaCl) with PIC. Then, 30 μg sonicated chromatin was incubated 7.5 μl of H3K27ac (Cell Signaling, 8173), H3K9ac (Cell Signaling, 9649), H3K36me3 (Cell Signaling, 9733) antibody or 5 μg H2A.Z (Abcam, ab4174) antibody with rotation at 4°C overnight. For each reaction, mix 25 μl Protein A and 25 μl Protein G dynabeads at 1:1 ratio and blocked with 1% BSA in ChIP dilution buffer overnight. Beads were washed 3 times with ChIP dilution buffer before added into the lysates. Incubated at 4°C for 4h with rotation. The beads were washed twice with RIPA-150 (50 mM Tris-HCl, pH 8.0, 157 150 mM NaCl, 1 mM EDTA, pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), then twice with RIPA-500 (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM EDTA, pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate). The beads were further washed twice with RIPA LiCl (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 1% NP-40, 0.7% sodium deoxycholate, 500 mM LiCl). The beads were washed twice with TE and resuspended with 150 μl freshly made elution buffer (0.1M NaHCO3, 1% SDS) with 5 μl Proteinase K and 2 μl RNase A (Thermo, R1253). Incubated at 56 °C overnight with shaking 900 rpm. The DNA library was prepared using a Next Ultra DNA Library Prep Kit (NEB, E7645) according to the manufacturer's instructions. The libraries were purified with AMPure XP beads (Beckman, A63881) according to the manufacturer 's instructions, and sequenced using Illumina platforms.