title: GSM4819684: 4C-seq Individual 12; Mus musculus; OTHER
library_construction_protocol: Immediately following dissection, C57BL/6J livers were snap frozen in liquid nitrogen and manually pulverised. ChIP-seq: Chromatin immunoprecipitation (ChIP) was performed as previously described with some modifications (Imbeault et al. 2017). 100mg of powdered frozen mouse liver was crosslinked in 1% formaldehyde for 10 minutes and subsequently quenched with Tris pH 8.0 (250mM final) for 10 minutes. The quenched cells were washed twice with ice-cold PBS supplemented with a protease inhibitor cocktail (EDTA-free cOmpleteTM, Sigma Aldrich), flash frozen in liquid nitrogen, and stored at -80°C. Crosslinked cells were thawed on ice and then lysed sequentially on ice and for 10 minutes at each step in each of the following buffers: LB1 (50 mM HEPES- KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton-X-100 and EDTA-free cOmpleteTM), LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and EDTA-free cOmpleteTM), and SDS shearing lysis buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.15% SDS and EDTA-free cOmpleteTM). The lysates were sonicated (Bioruptor) at 4°C to generate DNA fragments of 100 – 500 bp (3 repetitions of 5 sonication cycles of 30 seconds on and 30 seconds off) and the sonicated lysates were subsequently clarified by centrifugation (15,000 rpm for 15 min at 4 ̊C). The sonicated lysate was divided into 1.5-mL Eppendorf tubes based on the number of ChIPs performed and topped up to 1mL with Lysis Buffer 500NaCl (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton-X-100, 0.1% Sodium deoxycholate, 0.1% SDS and EDTA-free cOmpleteTM). This mixture was incubated overnight at 4°C with beads (Protein A Dynabeads, Invitrogen) that had been pre-blocked with 0.5% BSA and mixed with polyclonal CTCF antibody (C15410210-50 Diagenode). To remove non-specifically bound proteins, beads were washed five times with RIPA Buffer (50 mM HEPES-KOH, pH 7.4, 500 mM LiCl, 1 mM EDTA, 1% NP-40 and 0.7% Sodium deoxycholate) and once with TE Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA) at 4°C. The DNA-protein complex was eluted from the beads in Elution Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA and 1% SDS) for 20 min at 65°C, and reverse-crosslinked overnight at 65°C. The eluted samples were then treated with RNase A (Wako) and Proteinase K (Roche), and purified using a PCR purification kit (NEB). 4C-seq: 4C-seq was performed as previously described with some modifications (Splinter et al. 2012). Tissues were fixed as outlined in the ChIP protocol above. DpnII (New England Biolabs) was used as the primary restriction enzyme and NlaIII (New England Biolabs) as the secondary restriction enzyme. Prior to library preparation, samples were purified by the Monarch PCR & DNA Cleanup Kit (New England Biolabs). ChIP-seq: ChIP-seq libraries were prepared using KAPA Adapters, KAPA HyperPrep Kit (KAPA Biosystems), and AMPure XP Beads (Beckman Coulter) and quality checked using Qubit, Bioanalyzer, and Tapestation. The libraries were sequenced as 150bp paired-end reads on the Illumina HiSeq4000. 4C-seq: 16 individual PCR reactions were performed for each sample per viewpoint with reverse primers containing indexes. The 16 PCRs were combined and purified using 0.8x Agencourt AMPure XP beads (Beckman Coulter). Five libraries for each of the five tested viewpoints were multiplexed, quality checked using Qubit and Bioanalyzer, and sequenced as 150bp paired-end reads on the Illumina HiSeq4000.