Illumina HiSeq 2500 sequencing; HNF1A, H3K4me3 and H3K27ac ChIP-seq in control and Haster-deficient mouse livers
HNF1A, H3K4me3 and H3K27ac ChIP-seq in control and Haster-deficient mouse livers
Liver tissue was collected after perfusion of ice-cold PBS. Tissue was then minced with a razor blade in ice-cold PBS. Minced liver tissue (100 mg) or 100-500 mouse islets were incubated with 1% formaldehyde (Agar Scientific) for 10 min at RT. The reaction was stopped by addition of 1:10th of 1.25 M glycine for 5 min at RT, pelleted at 4°C for 3 min at 800g and washed twice with PBS. Crosslinked samples were lysed using ice-cold Lysis Buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8, 1 mM EDTA pH 8, 1x protease inhibitor cocktail) for 15-20 minutes on ice. Chromatin was sonicated using a S220 Focused-ultrasonicator (Covaris), with the following settings: 2% Duty Factor, 105W Peak Incident Power, 200 Cycles per Bust for a duration of 16 minutes. Sheared chromatin was centrifuged at full speed 10 min 4°C to remove debris and insoluble chromatin, and the supernatant was transferred to a fresh low-binding tube. For liver, chromatin equivalent to 5 µg of DNA was used for one histone mark ChIP and 10 µg for transcription factor ChIP. Chromatin was diluted 4 times with ChIP Dilution Buffer (0.75% Triton X-100, 0.1% Na-deoxycholate, 140 mM NaCl, 50 mM HEPES pH8, 1 mM EDTA, 1x protease inhibitor cocktail) and 5 % was used as input. 30 µL of Dynabeads Protein G (ThermoFisher) were blocked with BSA overnight at 4ºC. 10 µL of HNF1A antibody (Cell Signaling Technology, D7Z2Q), 2 µg of H3K27ac antibody (Abcam, ab4729) or 2 µg of H3K4me3 antibody (Merk, 15-10C-E4) were added to the samples and incubated overnight rotating at 4°C. 30 µl of magnetic beads were added to the samples and rotated at 4°C for 2h. Antibody-incubated samples were washed twice with RIPA-LS (10 mM Tris-HCl pH 8, 140 mM NaCl, 1 mM EDTA pH 8, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100), twice with RIPA-HS (10 mM Tris-HCl pH 8, 500 mM NaCl, 1 mM EDTA pH 8, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100), twice with RIPA-LiCl (10 mM Tris-HCl pH 8, 250 mM LiCl, 1 mM EDTA pH 8, 0.5% IGEPAL, 0.5% sodium deoxycholate) and once 10 mM Tris-HCl pH 8. Beads were resuspended in 20 µL of tagmentation solution (10 mM Tris-HCl pH 8, 5 mM MgCl2, 10% v/v dimethylformamide) containing 1 µL Tn5 (Illumina) and incubated at 37ºC for 10 min. The reaction was stopped by adding 1 mL ice-cold RIPA-LS buffer and incubated 5 min on ice. Beads were then washed twice with RIPA-LS, twice with TE, and resuspended in Elution buffer (10 mM Tris-HCl pH 8, 5 mM EDTA pH 8, 300 mM NaCl, 0.4% SDS). Proteinase K was added to the elution and incubated 1h at 55˚C, 1200 rpm, then overnight at 65˚C, 1200 rpm. DNA was purified using MinElute PCR Purification Kit (Qiagen). To estimate the number of cycles required for the amplification of the libraries, 2 µL of the elution was used for Syber Green qPCR, using Kapa HiFi polymerase (Kapa Biosystems). Libraries were amplified from 20 µL of elution with Kapa HiFi polymerase and Nextera primers, for Ct value roundup + 1 cycles. DNA clean-up was performed with 1.8x volume and size selection with 0.65x volume of AMPureXP beads (Beckman Coulter).