GSM8669068: Foxa1_Input_rep1; Mus musculus; ChIP-Seq
A total of 10⁷ cells were crosslinked for 8 minutes at room temperature in 10 mL PBS supplemented with 1% formaldehyde (Sigma, F8775). The reaction was quenched by adding glycine to a final concentration of 0.125 mM. Cells were pelleted by centrifugation at 500 × g for 3 minutes at 4°C, washed once with PBS, pelleted again, flash-frozen in liquid nitrogen, and stored until further processing. 30µL of a 1:1 mixture of Protein A/G Dynabeads (Invitrogen, 10002D and 10004D) was pre-coupled with 3 µL of FOXA1 antibody (ab170933) for 1 hour at room temperature, followed by three washes with 1× ChIP buffer and stored on ice until use. The cells were lysed in 10 mL of buffer A (50 mM HEPES pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) on ice for 10 minutes. After centrifugation, the pellet was resuspended in 5 mL of buffer B (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl) and incubated on ice for 5 minutes. Nuclei were pelleted by centrifugation at 500 × g for 3 minutes at 4°C, resuspended in 150 µL of buffer C (50 mM Tris pH 8.0, 5 mM EDTA, 1% SDS, 100 mM NaCl), and incubated on ice for 10 minutes. TE buffer (1.3 mL) was then added, and chromatin was sheared in 15 mL tubes using a Bioruptor Pico device for two sessions of 10 cycles (30 seconds ON / 30 seconds OFF). After shearing, 160 µL of 10× ChIP buffer (0.1% SDS, 10% Triton X-100, 12 mM EDTA, 167 mM Tris-HCl pH 8.0, 1.67 M NaCl) was added. The chromatin was transferred to fresh tubes and centrifuged at 13,000 × g for 10 minutes at 4°C. Per sample, 30 µL of 1:1 pre-coupled beads were added to the chromatin, and the mixture was incubated with rotation for 4 hours at 4°C. The ChIP samples were washed four times with LSB (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate), twice with HSB (same as LSB but with 500 mM NaCl), twice with LiSB (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate), and once with TE+ buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 50 mM NaCl). During the final wash, beads were transferred to fresh tubes, and the wash buffer was completely removed. DNA was eluted twice from the beads using 75 µL of elution buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 150 mM NaCl, 1% SDS) by incubation at 65˚C with shaking. The eluates were pooled, and 2 µL of RNase A (20 µg/µL) was added, followed by incubation for 1 hour at 37°C. Input samples were adjusted to a total volume of 150 µL with elution buffer and processed in the same manner as ChIP samples. Proteinase K (2 µL, 20 mg/mL) was added, and samples were incubated for 2 hours at 55°C and then for 6 hours at 65°C. Afterward, 9 µL of 5 M NaCl, 30 µL of AMPure XP beads, and 190 µL of isopropanol were added. Samples were vigorously mixed, incubated at room temperature for 10 minutes, and the beads were collected on a magnetic rack. The beads were washed twice with 80% ethanol, and DNA was eluted in 30 µL of 10 mM Tris-HCl pH 8.0 for 5 minutes at 37°C. For library preparation, 25 µL of ChIP DNA or 50 ng of input DNA was used with the NEBNext Ultra II Library Prep Kit for Illumina (NEB), following the manufacturer's protocol but with reactions scaled down to half-volume. Libraries were sequenced on the NovaSeq 6000 platform (Illumina) with paired-end reads (2x 56 bp).