GSM5348561: Hi-C_YMIIO_1; Mus musculus; Hi-C
Ovaries were separated and transferred into M2 medium (Sigma). For oocyte and GCs at growing stage (GO and GCs), the follicles with a diameter of 200-250 μm were isolated using insulin syringe under stereoscope. Full-grown oocytes and GCs (FGO and FGCs) were obtained 48 h after pregnant mare serum gonadotropin (PMSG) priming. For oocyte and GCs at MI stage (MIO and MIGCs), C57BL/6 female mice were intraperitoneally injected with 10 units PMSG, and 48 h later injected with 10 units human chorionic gonadotropin (hCG). After 9 h, the mice were sacrificed by cervical dislocation and the ovaries were separated. The pre-ovulatory follicles were punctured using a 1 mL disposable syringe under stereoscope, and COCs were transferred into new hyaluronidase droplets with the mouth pipette for 5 minutes of digestion. For oocyte and GCs at MII stage (MIIO and MIIGCs), the COCs were isolated from oviduct after 13 h and cumulus masses were separated in hyaluronidase medium. Oocytes and GCs from young (6 weeks old ) and aged (10 months old) mice at four stages were collected and processed for smart RNA-seq. Hi-C libraries of different cell types were prepared using a previously described in situ Hi-C protocol with minor modifications. Isolated oocytes were fixed with 920 μl 1% v/v formaldehyde in 1x HBSS at room temperature for 10 minutes. 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 and on ice for at least 15 min to quench crosslinking. The fixed cells were collected with oral siphon and washed twice in Hanks Buffer. Purified oocytes were lysed with 100 μl 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 μl 1x NEB Buffer 3.1, resuspended in 2 μl 10x NEB Buffer 3.1, 14 μl ddH2O, and 4 μl 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 μl ddH2O, and 1 μl (50 U) of DpnII restriction were added into the samples to bring total reaction volume to ~25 μl. Digestion was performed overnight at 37°C on a thermomixer with interval shaking (shake at 950 rpm 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 (3.75 μl of 0.4 mM biotin- 14-dATP (Life Technologies, 19524-016), 0.15 μl of 10 mM dCTP/dGTP/dTTP, 1 μl of 5 U/μl Klenow fragment (3'→5' exo-), 0.6 μl 10X NEB 3.1, 0.2 μl Milli-Q water) was added to the reaction and samples were incubated at 37°C for 1 hr in thermomixer (shake at 950 rpm for 10 sec with 5 min intervals). 90 μl of ligation master mix (49.8 μl of water, 24 μl of 5X T4 DNA ligase buffer (Invitrogen), 10 μl of 10% Triton X-100, 1.2 μl of 10mg/ml Bovine Serum Albumin (100X BSA), 5 μl 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 μl 1X NEB buffer 3 and resuspended with 50 μl 1X NEB buffer. To reverse crosslinking, 3 μl 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 130 μl 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 μl with Milli-Q H2O. To purify biotinylated DNA fragments, 50 μl Dynabeads MyOne Streptavidin T1 beads (Life technologies, 65602) were washed twice with 100 μl 1X Tween Washing Buffer (TWB, 5 mM Tris-HCl (pH 7.5); 0.5 mM EDTA; 1 M NaCl; 0.05% Tween 20), and then resuspended in 300 μl 2X Binding Buffer (10 mM Tris-HCl (pH 7.5); 1 mM EDTA; 2 M 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 100 μl 1X TWB on a Thermomixer at 55°C for 2 min with mixing and resuspended in 50 μl Milli-Q water. Samples were amplified with Phusion High-Fidelity DNA Polymerase (Thermo, F-530L) for 14-16 cycles. For size selection, 0.6X 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 0.2X 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 700 μl 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 25-50 μl of 1X Tris buffer. All Hi-C libraries were sequenced on Illumina Nova Seq 6000 platform at PE150 mode.