Pe pcr 1

Hi-C library generation was carried out as described previously^{22 (link)}. For each condition and each replicate, 20 million cells were fixed in 2% formaldehyde for 5 min, cells were incubated on ice for 30 min in 25 mL of ice-cold lysis buffer. After overnight digestion with HindIII at 37 °C, DNA ends were labeled with biotin-14–dATP (Life Technologies) using a Klenow end-filling reaction. Biotinylated DNA ends were then ligated together in an overnight ligation step using T4 DNA ligase (Invitrogen). After phenol: chloroform/ethanol purification DNA was quantified using Qubit, with a maximum of 40 μg taken forward. DNA was sheared to a peak concentration of around 400 bp, using the manufacturer’s instructions (Covaris). Sheared DNA was then end-repaired, polyadenine tailed, and double size selected using AMPure XP beads to isolate DNA ranging from 250 to 550 bp in size. Ligation fragments marked by biotin were immobilized using MyOne Streptavidin C1 DynaBeads (Invitrogen) and ligated to paired-end adapters (Illumina). Hi-C libraries were then amplified using PE PCR 1.0 and PE PCR 2.0 primers (Illumina) with eight PCR amplification cycles.

In nucleus, Hi-C library generation was performed as previously described [21 (link)]. Briefly, fixed adult liver tissue from three biological replicates at ZT0 0, 6, 12, and 18 was sieved through a 70-μM cell strainer and dounce homogenized in 10 ml of ice-cold lysis buffer with a tight pestle for a total of 30 strokes on ice. Nuclei were washed and permeabilized with 0.3% SDS for 45 min at 37 °C and then incubated overnight with HindIII at 37 °C, DNA ends were labeled with biotin-14-dATP (Life Technologies) in a Klenow end-filling reaction and ligated in nuclei overnight. DNA was purified by phenol-chloroform, and the concentration was measured using Quant-iT PicoGreen (Life Technologies). A total of 10 μg of DNA was sheared to an average size of 400 bp using a Covaris machine and following the manufacturer’s instructions. The sheared DNA was end-repaired, adenine tailed, and subject to a double size selection using AMPure XP beads to isolate DNA ranging from 250 to 550 bp in size. Ligated fragments marked by biotin-14-dATP were pulled-down using MyOne Streptavidin C1 DynaBeads (Invitrogen) and ligated to paired-end adaptors (Illumina). The in nucleus Hi-C libraries were amplified using the PE PCR 1.0 and PE PCR 2.0 primers (Illumina) using 6–9 PCR amplification cycles as required.