GSM4023463: Hi-C Ring1 Rnf2 dKO FGO rep2; Mus musculus; Hi-C
To isolate PGCs, germinal ridges were isolated from E11.5 and E13.5 embryos. To obtain the single cell su¬spension, the germinal ridges were digested with 0.25% trypsin-EDTA at 37°C for 10min. For E13.5 embryos, the germinal ridges were digested separately based on the gender. GFP-positive PGCs were sorted and collected by FACS.WT oocytes were collected from wild-type C57BL/6N females. Briefly, growing oocytes were isolated by mechanical dissection using a 26 gauge needle in M2 medium. Growing oocytes I were isolated from 7 or 10d old mice and growing oocytes II were from 14d old mice. Full-grown oocytes were isolated from superovulated 8-week-old mice 46 h after PMSG injection. The whole ovaries were clipped mechanically with a razor blade. To remove the granulosa cells, the dissociated oocyte-granulosa cell complexes were transferred into M2 medium containing 0.1% hyaluronidase and digested for 5 min. Oocytes were then crosslinked with 1% formaldehyde after removing the zona pellucida by acidic Tyrode's solution treatment. Eedfl/wt and EedmNull late 2-cell embryos were harvested by mating Eedfl/fl  or Eedfl/fl;Gdf9-Cre females with PWK/PhJ males. sisHi-C libraries were produced as described (Du et al., 2017). Briefly, PGCs, oocytes or embryos were cross-linked with 1% formaldehyde at room temperature (RT) for 10 min. Then formaldehyde was quenched with glycine for 10 min at RT. After being washed with 1 × PBS, the samples were incubated in lysis buffer (10mM Tris-HCl pH7.4, 10mM NaCl, 0.1mM EDTA, 0.5% NP-40 and proteinase inhibitor) on ice for 50 min and the chromatin was solubilized with 0.5% SDS. The nuclei were digested with MboI at 37 °C overnight. After fill-in with biotin-14-dCTP, the fragments were ligated in a small volume. Reversal of cross-linking, DNA purification, and sonication were done sequentially. The biotin-labeled DNA was then pulled down with Dynabeads MyOne Streptavidin C1 (Thermo Fisher, Cat # 65001). The fragments that included a ligation junction were subjected to Illumina library preparation. 9-15 cycles of PCR amplification were performed with Extaq (Takara), and the products were purified and size-selected with AMPure XP beads （Beckman Coulter, Cat # A63881）  RNA-seq libraries of Ring1-/-Rnf2fl/fl and Ring1/Rnf2 dKO oocytes were prepared as follows. Cumulus oocyte complexes were isolated from ovaries of approx. 3 months old females. Granulosa cells were removed by repeated pipeting using a fine-pulled capilary glass needle in M2 medium. Oocytes were washed twice in M2 medium and subsequently washed twice in DNA suspension Buffer (TEKnova, Cat # PNT0221). For RNA isolation, 15 GV oocytes from one mouse were pooled and RNA was extracted using the RNAEasy Micro Kit (Qiagen,Cat # 74004) according to the manufacturer's instructions. RNA was amplified and converted to cDNA using the Ovation v2 Kit (NuGen, Cat # 7102). Sequencing libraries were prepared with the Truseq DNA LT kit (Illumina, Cat # FC-121-2001).  CUT&RUN libraries of Eedfl/fl and Eed-/- oocytes were conducted as previously described (Skene and Henikoff, 2017). A low constant amount of yeast DNA was added to each reaction as spike-in DNA.
GEO Accession
GSM4023463