title: GSM8651781: ChIL-seq sample for Hnf4a in iHepC, replicate 2; Mus musculus; OTHER
library_construction_protocol: ChIL-seq was performed following a modified protocol of the original ChIL-seq (PMID: 30532068). 10,000 cells were plated on typeⅠcollagen coated 96-well plates and let to attach for 16 h in culture. Cells were then fixed with 1% formaldehyde in PBS for 5 min and permeabilized with 1% Triton X-100 in PBS for 20 min. Samples were incubated in 10% Blocking One-P (Nacalai Tesque) in PBS with anti-Hnf4α mAb (Cell Signaling Technology; #3113) for 6 h at room temperature, washed three times in PBS and then incubated at 4℃ overnight in 10% Blocking One-P in PBS containing 500 mM NaCl and the ChIL probe at 2 μg/ml. After washing in PBS, samples were incubated in 50 µl Tn5 solution [50 mM HEPES-KOH (pH 7.2), 0.1 M NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton X-100 and 10% glycerol] containing 0.5 µl Tn5 transposase (given by Dr. Yasuyuki Ohkawa) for 10 min at room temperature. Tn5-MEDS-B oligo (2 µM) was then added at 0.5 µl per well in 1× dialysis buffer [50 mM HEPES-KOH (pH 7.2), 0.1 M NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton X-100 and 10% glycerol] and incubated for 60 min at room temperature. Samples were then washed in 1× dialysis buffer and three times in PBS, incubated in 100 µl 1× TAPS-DMF buffer [10 mM TAPS-NaOH (pH 8.5), 5 mM MgCl2 and 10% N,N-dimethylformamide] for 1 h at 37℃, and washed at three times in PBS. To stop the Tn5 reaction, cells were incubated in 100 µl 0.2% SDS for 10 min at room temperature. After washing at three times in PBS, samples were incubated in 50 µl reaction mixture [200 U T4 DNA ligase (New England Biolabs), and 1.5 U T4 DNA polymerase (New England Biolabs), in 1× T4 DNA ligase reaction buffer containing 0.1 mM dNTP] for 30 min at room temperature. After discarding the solution, 100 µl 0.2% SDS were added to remove DNA-bound enzymes, followed by incubation for 10 min at room temperature and three washes in PBS. In situ transcription was then carried out using T7 RNA polymerase (200 U per well; Ambion) for 20 h at 37 ℃. The samples were treated with DNase I (2.5 U per well; Thermo Fisher Scientific) for 30 min at 37℃ and RNA was extracted using a RNAClean XP beads (Beckman Coulter). Purified RNA was reverse-transcribed with SMARTScribe reverse transcriptase (Takara) following the manufacturer's instructions and the resulting cDNA was amplified by SeqAmp DNA Polymerase (Takara) by 14 cycles. After amplification, small fragments (<150 bp) were removed and large size of fragments (>600 bp) were removed using AMPure XP beads (Beckman Coulter). The amplified DNA was purified using AMPure XP beads and the quality of the libraries was assessed using a Bioanalyzer 2100 (Agilent Technologies). Samples were paired-end sequenced at PE100 on an Illumina NovaSeq 6000 platform. As detailed described in previous report (PMID: 30532068).