GSM8746579: DLD1-TetOff-flag-Cdx2 (ChIP); Homo sapiens; ChIP-Seq
Twenty hours after induction, the cells were fixed by adding 540 µL of formaldehyde (a final concentration, 1%; 064-03843; FUJIFILM) to 20 mL of the culture medium and incubated for 5 min at 25 °C. Glycine (10x, 2mL; 077-00735; FUJIFILM) was added to the ten dishes, which were gently rotated for 5 min at 25 °C. The cells were washed twice with 10 mL of ice-cold phosphate-based saline (PBS) and scraped with 1 mL of ice-cold PBS. Cells from the ten dishes were pooled into a 15-mL centrifuge tube and centrifuged at 3,500 rpm (2,380 g; AX-320, TOMY) for 3 min at 4 °C, after which the supernatant was then discarded.  According to the manufacturer's instructions of the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads; 9004; Cell Singnaling Tech.), nuclei were prepared and treated with 0.5 µL of micrococcal nuclease solution per sample at 37 °C for 50 min (MNase; Worthington Bio. Corp.). The MNase digestion reaction was terminated by adding 10 µl of 0.5 M ethylenediaminetetraacetic acid (EDTA). The length of the digested DNA (approximately 150 bp) was confirmed via agarose gel electrophoresis. The samples were centrifuged at 13,000 rpm (15,300 g; MX-307, TOMY) for 1 min at 4 °C, and the supernatant was discarded. The cell pellet was re-suspended in 100 µL of a ChIP dilution buffer (150 mM NaCl, 20 mM Trsi-HCl, 1% Triton X-100, and 2 mM EDTA), incubated for 10 min on ice, transferred to TPX tubes, and sonicated to disrupt the nuclear membrane at 4 °C using a Biorupter II (Sonicbio). The samples were then centrifuged at 10,000 rpm (9,100 g; MX-307, TOMY) for 10 min at 4 °C, and the supernatant was transferred to a new tube. A 19-fold volume of the ChIP dilution buffer was added to the digested chromatin, and the mixture was incubated with anti-flag M2-antibody-conjugated agarose beads (A2220; Sigma Aldrich) for 12 h at 4 °C with rotation. The beads were washed six times with ChIP dilution buffer and treated with an RNase (final concentration, 20 µg/mL; Worthington Bio. Corp.) for 1.5 h at 4 °C. To elute the flag-Cdx complex, the beads were incubated with 200 µL of the ChIP dilution buffer containing the 3 x FLAG peptide (F4799; Sigma Aldrich) at 37 °C for 1 h. The samples were centrifuged at 15,000 rpm (20,400 g; MX-307, TOMY) for 1 min at 4 °C, and 180 µL of the supernatant was transferred to a new tube. A 2 x elution buffer of 2% sodium dodecyl sulfate (SDS) and 0.2 M NaHCO3 was added to the samples, and the mixtures were incubated at 65 °C overnight. The DNA was purified by the standard phenol-chloroform extraction method. ChIP-seq libraries were prepared using the Illumina TruSeq ChIP Sample Prep Kit following manufacturer's protocols.