title: GSM5253085: Control_ChIP Technical 1; Homo sapiens; ChIP-Seq
library_construction_protocol: 20 million SNU-398 cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature. The reaction was terminated by adding 2M glycine to a final concentration of 125mM. Cells were then washed with 1´PBS and resuspended in 1mL of cell lysis buffer (20mM Tris pH8.0, 85mM KCl, 0.5% nonidet P-40, protease inhibitor). After 10 minutes of incubation on ice, cells were spun down and cell pellet was resuspended in another 1mL of cell lysis buffer. After another 5 minutes of incubation on ice, cells were spun down and cell pellet was resuspended in 1mL of nuclear lysis buffer (10mM Tris-HCl pH7.5, 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor). After 10 minutes of incubation on ice, chromatin was sheared to 500bp. Antibody‑protein A/G Dynabead conjugate was prepared by adding 0.75µg of SALL4 rabbit monoclonal antibody (Cell Signaling Technology #8459) to pre-washed 50µL of protein A/G Dynabeads (Life Techonlogies) with one hour incubation at 4°C with rotation. Sheared chromatin was then added to antibody-protein A/G conjugate and incubated overnight at 4°C with rotation. After overnight incubation, the beads were washed sequentially with the following buffers: twice with RIPA/500mM NaCl buffer (0.1% deoxycholate, 0.1% SDS, 1% Triton X-100, 500mM NaCl, 1mM EDTA, 20mM Tris-HCl pH8.1), twice with LiCl buffer (0.25M LiCl, 1% nonidet P-40, 1% sodium deoxycholate, 1mM EDTA, 10mM Tris-HCl pH8.1), twice with TE buffer (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0). Protein complexes were reverse cross-linked with 50µL of ChIP Elution Buffer (10mM Tris-HCl pH8.0, 5mM EDTA, 300mM NaCl, 0.1% SDS) and 8µL of Reverse Crosslink Mix (250mM Tris-HCl pH6.5, 1.25M NaCl, 62.5mM EDTA, 5mg/mL proteinase K, 62.5µg/mL RNase A) at 65°C for 5 hours. Reverse cross-linked DNA was cleaned up using SPRI beads (Beckman Coulter) and eluted in 10mM Tris-HCl pH 8.0. To generate libraries for deep sequencing, the eluted DNA was end‑repaired using End-It DNA End-Repair Kit (Epicenter #ER0720) and A-tailing was then carried using Klenow (3'-5' exo-) enzyme (New England Biolabs). Illumina sequencing adaptors were ligated to the DNA fragments and adaptor-ligated DNA fragments were enriched with 14 cycles of PCR. DNA libraries were gel purified and analyzed on Bioanalyzer (Agilent) for their size distribution. Libraries were sequenced on Illumina HiSeq 2500 sequencer with single-end 35bp settings.