title: GSM4718257: PreL_1_Hi-C; Mus musculus; Hi-C
library_construction_protocol: Murine spermatocytes were isolated by FACS as described previously (Gaysinskaya, Soh, van der Heijden, & Bortvin, 2014; Getun, Torres, & Bois, 2011) with modification. Wild-type or mutant mice of different ages were used for isolation of spermatocytes at different stages: 7-11 days old mice were used for isolating Sertoli, spermatogonia, and pre-leptotene cells,  2-week old mice for zygotene cells, 5-week old mice for pachytene, diplotene, and Metaphase II cells, and 1-2 months old SUN1 W151R mutant mice for zygotene-like spermatocytes Hi-C libraries of different cell types were prepared using a previously described in situ Hi-C protocol (Rao et al., 2014) with minor modifications. 0.1 to 1 million cells were fixed with 920 ul 1% v/v formaldehyde in 1x HBSS at room temperature for 10 minutes with mixing. 2.5M Glycine was added to the cells to make the final concentration of Glycine 0.2 M. Cells were incubated at RT for 5 min with mixing and on ice for at least 15 min to quench crosslinking. The fixed cells were centrifuged at 3000xg for 10 min at 4°C. The supernatant was discarded and cell pellets were resuspended with 100 ul ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630) with protease inhibitors. Samples were incubated on ice for 15 minutes and centrifuged for 5 minutes at 3,000xg at 4°C to isolate nuclei. The nuclei pellets were washed once with 100 ul 1x NEB Buffer 3.1, resuspended in 2 ul 10x NEB Buffer 3.1, 14 ul ddH2O, and 4 ul of 0.5% SDS and then incubated at 65°C for 5 min to open up chromatin. 2.2 μl of 10% Triton X-100 (Sigma, 93443) was added to the samples, and samples were incubated at 37°C for 15 minutes to quench SDS. To digest chromatin, 0.5 μl of 10X NEB 3.1 Buffer, 1.5 ul ddH2O, and 1 ul (50U) of DpnII restriction were added into the samples to bring total reaction volume to ~25 ul.  Digestion was performed overnight at 37°C on a thermomixer with interval shaking (shake at 950rpm for 10 sec with 5 min intervals). Following digestion, samples were incubated at 65°C for 20 min to inactivate the restriction enzyme, then cool to RT on ice. To fill the restriction overhangs, 6μl of fill-in master mix (3.75μl of 0.4mM biotin-14-dATP (Life Technologies, 19524-016), 0.15μl of 10mM dCTP/dGTP/dTTP, 1μl of 5U/μl Klenow fragment (3' → 5' exo-), 0.6μl 10X NEB 3.1, 0.2μl Milli-Q water) was added to the reaction and samples were incubated at 37°C for 1 hr in thermomixer (shake at 950rpm for 10 sec with 5 min intervals). 90 ul of ligation master mix (49.8μl of water, 24μl of 5X T4 DNA ligase buffer (Invitrogen), 10μl of 10% Triton X-100, 1.2μl of 10mg/ml Bovine Serum Albumin (100X BSA), 5μl of 1 U/ μl T4 DNA Ligase (Invitrogen)) was then added and samples were mixed by inverting and incubated at 16 °C for 4 hours. After ligation, samples were centrifuged for 5 min at 3,000g. The pellets were washed with 100 ul 1X NEB buffer 3 and resuspended with 50 ul 1X NEB buffer. To reverse crosslinking, 3ul 20mg/ml proteinase K (NEB, P8102) and 5μl of 10% SDS were added into the samples and then incubated at 65°C for 2hrs. Phenol-chloroform extraction and ethanol precipitation were performed. The purified DNA was dissolved in 130ul 1x Tris Buffer and sheared by Covaris M220(Peak Incident Power: 50W; Duty factor: 20%; Cycles per Burst: 200; Treatment Time: 65s). To minimize sample loss, size selection was not performed after shearing. End repair of DNA fragments and ligation with Illumina Truseq adapters was performed using End Repair/dA-Tailing Module (NEB, E7442L) and Ligation Module (NEB, E7445L) according to product manuals. Following adaptor ligation, the total volume of DNA samples was brought to 300 ul with Milli-Q H2O. To purify biotinylated DNA fragments, 50μl Dynabeads MyOne Streptavidin T1 beads (Life technologies, 65602) were washed twice with 100μl 1X Tween Washing Buffer (TWB, 5mM Tris-HCl (pH 7.5); 0.5mM EDTA; 1M NaCl; 0.05% Tween 20), and then resuspended in 300μl 2X Binding Buffer (10mM Tris-HCl (pH 7.5); 1mM EDTA; 2M NaCl) before being added to DNA samples. The samples were incubated at RT for 15 minutes with rotation to bind biotinylated DNA to the streptavidin beads. Beads were washed twice with 100μl 1X TWB on a Thermomixer at 55°C for 2 min with mixing and resuspended in 50ul Milli-Q water. Samples were amplified with Phusion High-Fidelity DNA Polymerase (Thermo, F-530L) for 4-7 cycles. For size selection, 0.5X volumes of AMPure XP beads (Beckman Coulter, A63881) prewarmed to RT was added to the PCR products and samples were incubated at RT for 5 minutes. Samples were then separated on a magnet. The clear solution was mixed with another 0.5X volumes of AMPure XP beads by pipetting and incubated at RT for 5 minutes.  Samples were again separated on a magnet and the clear solution was discarded. Beads were washed once with 700μl freshly made 70% ethanol without mixing. After removing ethanol, tubes containing beads were left on the magnet for 5 minutes with lids open to allow the remaining ethanol to evaporate completely. DNA was eluted from beads with 25-50μl of 1X Tris buffer. All Hi-C libraries were sequenced on Illumina Nova Seq 6000 platform at PE150 mode.
attributes.tag: GEO Accession
attributes.value: GSM4718257