GSM3729089: Hi-C-prometa-rep1; Mus musculus; Hi-C
5million pro-meta or ana/telo cells and 10million interphase cells were collected from cell sorting per experiment. Cells were pelleted, re-suspended in 1ml cold Cell Lysis Buffer (10mM Tris pH 8, 10mM NaCl, 0.2% NP-40/Igepal) and incubated on ice for 10min. Cell nuclei was pelleted at 4℃ and washed with 800ul 1.2xDpnII buffer. Nuclei was pelleted at 4℃ and re-suspended in 500ul 1.2xDpnII buffer. 15.5ul SDS (10%) was added into the system to reach a final concentration of 0.3%. Samples were then incubated at 37℃ on a thermomixer for 1h with shaking at 950rpm. 50ul Triton X-100 (20%) was added to the system to reach a final concentration of 1.8%. Samples were further incubated at 37℃ on a thermomixer for 1h at 950rpm. 300U of DpnII restrictive enzyme (NEB, R0543M) was then added into the system and samples were incubated at 37℃for overnight with shaking at 950rpm. Another 300U of DpnII was added into the system and samples were further incubated at 37℃ for 2-4h with shaking at 950rpm. Samples were then incubated at 65℃ for 20min with shaking at 950rpm to inactivate DpnII. Nuclei was pelleted, cooled down to room temperature, re-suspended in 300ul 1xNEB buffer2, containing 50uM biotin-14-dATP (Thermal Fisher Scientific, 19524016), dGTP, dCTP, dTTP (Promega, U121B, U122B, U123B) and 40U DNA Polymerase I, Large (Klenow) fragment (NEB, M0210) and incubated at 37℃ for 1.5h with shaking at 950rpm. The system was then added with 120ul 10xT4 DNA ligase buffer, 12ul 100xBSA, 758ul nuclease free water and 10ul (4000U) T4 DNA ligase (NEB, M0202M). Ligation was carried out at 16℃ for 4h and room temperature for additional 2h. 20ul of 20mg/ml protease K (3115879 BMB) and 120ul of 10%SDS were added into the system and samples were de-crosslinked at 65℃ for overnight. An additional 10ul of protease K was added and samples were further de-crosslinked at 55℃ for 2h. DNase-free RNase was added into the system and samples were incubated at 37℃ for 30min. Ligated DNA was extracted with phenol chloroform extraction and re-constituted in 180ul nuclease free water. DNA concentration was quantified with a Qubit 4 Fluorometer (Thermal Fisher Scientific, Q33226). Ligated DNA was sonicated to 200-300bp (Epishear, Active Motif, 100% amplitude, 30s ON and 30s OFF, 25-30min) and cleaned up with AMPure XP beads (Beckman Coulter). Each sample of biotinylated DNA was pulled down with 100ul Dynabeads MyOne Streptavidin C1 beads (Thermal Fisher Scientific, 65002) by incubating at room temperature for 15min. The amount of bound DNA was calculated by subtracting the amount of unbound DNA from the amount of total DNA. DNA bound streptavidin beads were washed once with 1xbead wash buffer (5mM Tris-HCl (pH 7.5), 0.5mM EDTA and 1M NaCl) and once with T4 DNA ligase buffer. Streptavidin beads were re-suspended with 84ml water. Library was prepared using NEBNext DNA Library Prep Master Mix Set for Illumina (NEB E6040, M0543L, E7335S) according to manufacture instruction. On-bead end repair was performed by mixing 84ul biotinylated beads with 10ul NEBNext End Repair Reaction Buffer (10x), 5ul NEBNext End Repair Enzyme Mix and 5U DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210). The mixture was incubated at room temperature for 30min. Beads with end repaired blunt DNA was washed once with 600ul 1xbead wash buffer and then 500ul 1xNEB buffer2. Beads was re-suspended in 42ul nuclease free water. Beads were then mixed with 5ul NEBNext dA-tailing reaction buffe (10x) and 3ul Klenow Fragment (3'→5' exo-) for dA-tailing. The mixture was incubated at 37℃ for 30min. Beads were then washed once with 600ul 1xbead wash buffer and then 600ul 1xT4 DNA ligase buffer.  Beads were re-suspended in 20ul nuclease free water. Adaptor ligation was performed by mixing the beads with 10ul Quick Ligation reaction buffer (5x), 15ul NEBNext Adaptor (15uM) and 5ul Quick T4 ligase. The mixture was incubated at room temperature for 15min. 3ul of USER enzyme was then added into the mixture and incubated at 37℃ for 15min. Beads were washed twice with 600ul 1xbead wash buffer and re-suspended in 50ul nuclease free water containing 0.1% SDS. Beads were incubated at 98℃ for 10min to release DNA and DNA was cleaned up with AMPure XP beads. Purified adaptor ligated DNA was then index labeled with NEBNext multiplex oligos for 6 cycles on a thermal cycler. PCR products were purified using AMPure XP beads. Sequencing was performed on an Illumina NextSeq 500 platform using Illumina sequencing reagents according to manufacturer's instructions.