GSM8634273: wapl_smc2_no_a_pmpm2_high_clone9; Mus musculus; Hi-C
For Hi-C, FACS purified cells (0.1 million) were lysed in 1ml cold Cell Lysis Buffer (10mM Tris pH 8, 10mM NaCl, 0.2% NP-40/Igepal) for 10min on ice. Nuclei were pelleted at 4℃ and washed once with cold 1.4 × NEB buffer 3.1 (NEB Cat#B7203S). Nuclei were then re-suspended in 25ul of 1.4 × NEB buffer 3.1 and treated with 0.1% SDS for 10min at 65℃. Nuclei were then treated with 1% Triton X-100 to quench SDS for 1h at 37℃. Chromatin was digested with 25U DpnII restriction enzyme (NEB, Cat#R0543M) at 37℃ overnight with shaking (950rpm on a thermal mixer). After overnight digestion, an additional stroke of 25U DpnII was added into the system to further digest the genome for 4 hours at 37℃. After the 2nd stroke of digestion, nuclei were incubated at 65℃ for 20min to inactivate DpnII. Digested chromatin fragments were then cooled down to room temperature and blunted with dCTP, dGTP, dTTP (Diamond Cat#B110049, B110050, B110051) and Biotin-14-dATP (active motif, Catalog No: 14138) using 6.6U DNA Polymerase I, Large (Klenow) fragment (NEB, Cat#M0210). Chromatin was then ligated in-situ with 2000U T4 DNA ligase (NEB, Cat#M0202M) for 4 hours at 16℃ followed by further incubation for 2 hours at room temperature. Chromatin was then reverse-crosslinked overnight at 65℃ in the presence of 1% SDS and proteinase K (G-clone Cat#EZ0970). After reverse-crosslinking, DNase-free RNase A (Vazyme Cat#DE111-01) was added to the system for 30min at 37℃ to remove RNA. DNA was then extracted by phenol-chloroform extraction, precipitated, and dissolved in 130ul of nuclease free water. For ChIP-seq, FACS purified cells were re-suspended in 1ml cold Cell Lysis Buffer (10mM Tris pH 8, 10mM NaCl and 0.2% NP-40) supplemented with 1:500 protease inhibitor and 1:100 PMSF for 20min on ice. Notably, for Rad21 ChIP-seq experiments in the WapldTag/SororinmAID and WapldTag/SMC2mAID/SororinmAID cells, roughly 30% of crosslinked 293T cells were added into the cell mixture. Nuclei were then pelleted and re-suspended in 1ml Nuclear Lysis Buffer (50mM Tris pH 8, 10mM EDTA, 1% SDS, fresh supplemented with PI and PMSF) for 20min on ice. Chromatin was then fragmented by sonication (Qsonica Q800R3, 80% amplitude, 20s ON, 40s OFF) for 17min. Cell debris were removed by centrifugation at 15000g for 10min at 4℃. The supernatant was collected and diluted with 4 volumes of IP dilution buffer (20mM Tris pH 8.0, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS) supplemented with protease inhibitor, PMSF and 50ul of A/G agarose beads (Santa Cruz Cat#sc-2003). Preclear of chromatin was performed at 4℃ for 4-8h with rotation. Precleared chromatin was then incubated with 35μl A/G agarose beads (A:G = 1:1) pre-bound with antibody and incubated on a rotator at 4℃ overnight. Beads were washed once with IP wash buffer I (20mM Tris pH 8, 2mM EDTA, 50mMNaCl, 1% Triton X-100, 0.1% SDS), twice with high salt buffer (20mMTris pH 8, 2mM EDTA, 500mMNaCl, 1% Triton X-100, 0.01% SDS), once with IP wash buffer II (10mMTris pH 8, 1mM EDTA, 0.25 M LiCl, 1% NP-40/Igepal, 1% sodium deoxycholate) and twice with TE buffer (10mM Tris pH 8, 1mM EDTA pH 8). All washing steps were performed on ice. Beads were moved to room temperature and DNA was eluted in 200μl fresh Elution Buffer (100mM NaHCO3, 1%SDS). ChIP and input chromatin were reverse crosslinked at 65℃ overnight in the presence of proteinase K. IP and input samples were supplemented with 10ul of 3M sodium acetate (pH 5.2), and DNA was purified with PCR purification kit (QIAGEN Cat#28106). For Hi-C, DNA was sonicated to 200-300bp fragments with a QSonica Q800R3 sonicator (40% amplitude, 15s ON and 15s OFF, 13min sonication) and purified with VAHTS DNA Clean Beads (Vazyme Cat#N411). Biotin-labeled DNA fragments was enriched by incubation with 100μl Dynabeads MyOne Streptavidin C1 beads (Thermal Fisher Scientific, Cat#65002) at room temperature for 15min. Streptavidin C1 beads were then washed twice with 1 × B&W buffer (5mM Tris-HCl, pH 7.5; 0.5mM EDTA; 1M NaCl). Beads were re-suspended in 30ul of 1 × NEB Buffer2 (NEB Cat#7002S) containing 9U of T4 DNA polymerase (NEB, Cat#M0203S) and 40uM dATG/dGTP and incubated at 20℃ for 4 hours to remove unligated fragments. Beads were then washed twice with 1 × B&W buffer and subject to library construction. DNA end repair, dA-tailing and adaptor ligation were constructed on beads, using the AHTS Universal DNA Library Prep Kit for Illumina or MGI (Vazyme, Cat# ND627-01, NDM607-02) based on the manufacturer's protocol. To elute DNA from streptavidin beads, the library mixture was treated with 0.1% SDS and incubated at 98℃ for 10min. Released DNA was collected from the supernatant and purified with VAHTS DNA Clean Beads. Finally, the library was amplified for 9 cycles on a thermal cycler, using the VAHTS® HiFi Amplification Mix. PCR products were then purified with VAHTS DNA Clean Beads and sequenced on an Illumina NextSeq500 or MGI DNBSEQ-T7 sequencing platform. For ChIP, libraries were constructed using the VAHTS Universal DNA Library Prep Kit for MGI (Vazyme, Cat#NDM607-02) according to the manufacturer's protocol. ChIP libraries were sequenced on the MGI DNBSEQ-T7 sequencing platform. HiC and ChIP-seq libraries were constructed using the VATHS DNA library preparation kit from Vazyme (N607) according to the manufacturer's protocol.