title: GSM9301421: hKLF6 Invitrogen Antibody ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were spun down at 500 x g for 5 minutes in a refrigerated eppendorf centrifuge and crosslinked at a concentration of 10 million cells per 5 mL in freshly made 1x phosphate buffered saline without magnesium or calcium with formaldehyde-containing buffer (50 mM HEPES-KOH, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 8.8% formaldehyde in 10% FBS RPMI) added at 1/11 ratio to cell suspension. Crosslinking performed by incubating for 3 minutes on ice. After the crosslinking reaction, a 2.5 M glycine solution was added to make a final concentration of 125 mM, which were incubated at room temperature for 5 minutes to stop fixation. Cells were pelleted at 2000 x g for 10 minutes in a refrigerated eppendorf centrifuge, the formaldehyde-glycine supernatant was removed, and the cells were washed (resuspended and then pelleted at 2000 x g for 10 minutes in a refrigerated eppendorf centrifuge) in cold PBS twice. The fixed cell pellets were stored at -80 °C until the ChIPmentation protocol was performed. The fixed cell pellet was resuspended in 5 mL of L1 buffer (50 mM HEPES-KOH, pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 5 mM sodium butyrate and 1:100 dilution of 1 tablet/1 mL protease inhibitor (P8340; Sigma) and rotated at 4 °C for 10 minutes to lyse the cells. The cell solution was centrifuged at 2000 x g for 8 minutes in a refrigerated table-top centrifuge, the supernatant was removed, and the cell pellet was subjected to nuclei isolation from cytoplasm via incubation with 5 mL L2 buffer (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA, 5 mM sodium butyrate, and 1:100 dilution of 1 tablet/mL  protease inhibitor (P8340; Sigma) with rotation for 10 minutes at room temperature (RT). The cell solution was centrifuged at 2000 x g for 8 minutes in a refrigerated table-top centrifuge, the supernatant was removed, and the isolated nuclei pellets were carefully washed twice and resuspended in a 1X Tris-EDTA (TE) + 0.1% NP40 0.1% Triton X100 solution. The cells were sonicated (peak power 175.0, duty factor 10.0, cycle/burst 200) for 300 s in large 1 mL microtubes at 4 °C using a S220 focused ultrasonicator (Covaris) to obtain the 200- to 500-bp fragments of chromatin. The ChIP procedure was performed by the Diagenode IP-Star Compact automation system.Antibodies against KLF6 (2 µg; 39-6900; Invitrogen or AF3499; R&D systems) were bound to 4.5 uL of Protein G Dynabeads were incubated with 2 µg of chromatin for 1 hour in PBS + 0.04% NP-40 followed by sequential washes of with RIPA-150 buffer 1 (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, and 1% Triton X-100 in DI water), RIPA-300 buffer 2 (10 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, and 1% Triton X-100 in DI water), LiCl buffer 3 (250 mM LiCl, 0.5% sodium deoxycholate, and 0.5% NP-40 in TE buffer), and Wash buffer 4 (TE + 0.2% Triton X-100). Samples were eluted in 10 mM Tris-HCl containing 0.25 M NaCl to be processed via ChIPmentation. Washed Dynabeads were processed with loaded Tn5 transposase (C01070012; Hologic Diagenode) per an established ChIPmentation protocol to accomplish tagmentation of ChIPed DNA (Schmidl et al., 2015). The tagmented ChIPed DNA were washed with cold 10 mM Tris-HCl and incubated with proteinase K in elution buffer (TE with 250 mM NaCl and 0.3% SDS) for a cycle of 15 minutes at 42 °C, 4 hours at 60 °C, and 10 minutes at 15 °C. Lastly, the tagmented ChIPed DNA was purified from beads using the Qiagen MinElute DNA kit per the manufacturer's instructions. The library was amplified using a cycle number based on side reaction to stop amplification prior to saturation. The product was cleaned with AMPure beads and the size and distribution were checked on TapeStation (Agilent).