Illumina HiSeq 2500 paired end sequencing; UMI-4C in control and Haster-deficient mouse livers
UMI-4C in control and Haster-deficient mouse livers
Liver tissue from three samples per genotype were collected and crosslinked with 2% formaldehyde for 10 min. Frozen pellets of ~10e7 cells were thawed on ice and resuspended in 5 mL of Cold Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% IGEPAL, 1x protease inhibitor cocktail). After isolation, the nuclei were resuspended in 650 µL of nuclease-free water, 60 µL DpnII buffer, 15 µL 10% SDS and incubated at 37ºC, 1h, 900 rpm, with an additional hour after addition of 75 µL of 20% Triton X-100. The chromatin was digested at 37ºC, 900 rpm, for 24 hr using 600U of DpnII (R0543L, NEB), and enzyme was inactivated by incubating at 65ºC for 20 min. Ligation was performed in a final volume of 7 mL with 60 U of T4 DNA ligase (Promega) and incubated at 16ºC overnight. The efficiency of the digestion and ligation was assessed by gel electrophoresis. Chromatin was reverse crosslinked with 30 µL Proteinase K (10 mg/mL), overnight at 65ºC, followed by 45 min of incubation with 30 µL RNaseA (10 mg/mL) at 37ºC. The DNA was purified by phenol-chloroform extraction followed by ethanol precipitation, and resuspended in 10 mM Tris-HCl pH 8. 10 µg of DNA was sonicated using a S220 Focused-ultrasonicator (Covaris), to obtain 400-600 bp fragments. DNA was end-repaired with 10 µL NEBNext End Repair Mix (NEB, E6050L) in 200 µL final volume, incubated 30 min at 20ºC, purified with 2.2x AmpureXP beads (Beckman Coulter), and eluted in 10 mM Tris-HCl pH 8. A-tailing was performed with 200 U Klenow fragment (NEB, M0212M) in 100 µL 1X NEBbuffer 2 with 1nM dATP. 5' ends were dephosphorylated at 50ºC for 60 min with 20U calf intestinal alkaline phosphatase (NEB, M0290S). The DNA was then cleaned with 2x AmpureXP beads. Adapters were ligated with 0.4 µM Illumina-compatible forked indexed adapters and 10 µM quick ligase (NEB, M2200) in 160 µL 1x quick ligase buffer (NEB, M2200) for 15 min at 25ºC. DNA was denatured at 95ºC 2 min and cleaned with 1x AmpureXP beads. To generate UMI-4C libraries, two nested PCRs were performed using Go Taq polymerase (Promega) with 0.4 mM final concentration of primers. The 1st PCR used the upstream bait primer and the Illumina universal primer 2, and amplification was performed for 20 cycles. The DNA was cleaned with 1x AmpureXP and used for the 2nd PCR with the downstream bait primer and the Illumina universal primer 2 for 16 cycles. After the 2nd PCR, the DNA was cleaned, and size selected with 0.7x AmpureXP beads. The size distribution of the libraries was controlled by Bioanalyzer and the libraries were quantified by qPCR with KAPA library quantification kit (Roche, 07960166001).