title: GSM8767148: Chicken oocyte post-LBC stage nucleus, Hi-C, single nucleus, biol rep 1; Gallus gallus; Hi-C
library_name: GSM8767148
library_construction_protocol: Chicken oocytes, with diameters ranging from 1 to 2 mm as lampbrush chromosome and from 4 to 7 mm as post-lampbrush chromosome stages, were dissected from the ovary of healthy chicken and placed in individual drops of cooled "5:1" medium (83 mM KCl, 17 mM NaCl, 6.5 mM Na2HPO4, 3.5 mM KH2PO4, 1 mM MgCl2, 1 mM DTT, pH 7.2). All animal experiments were approved by the bioethics committee of the Institute of Cytology and Genetics SB RAS (Protocol №66, October 9th, 2020). International guidelines were followed during experimental procedures (“Guide for the Care and Use of Laboratory Animals” 97). Nuclei were isolated as described earlier (44), washed with "5:1" medium, and transferred to a dish containing 2% formaldehyde in PBS for fixation at room temperature for 15 minutes. The formaldehyde was quenched by adding glycine to a final concentration of 0.125 M for 15 minutes at room temperature. The integrity of the nuclei was assessed using an Olympus stereomicroscope, and the Hi-C protocol was initiated immediately. Individual fixed nuclei were transferred to microplate wells containing 9 μL of ice-cold lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal) for 20 minutes. The nuclei were then washed with NEB3.1 + 0.5% SDS and transferred to 0.2 mL PCR tubes with 3 μL of NEB3.1 + 0.5% SDS. 10 μL freshly prepared 1.2% Low Melting Point Agarose were added to each tube under a stereomicroscope. After solidification, 10 μL of NEB3.1 + 0.6% SDS was added, and the samples were incubated at 37 °C for 1 hour. SDS was quenched using 10 μL of 6% Triton X-100, and chromatin fragmentation was performed with 25 U of DpnII at 37 °C overnight. The digested chromatin ends were labeled using 5 U of the Klenow fragment in the presence of biotin-15-dCTP at 22 °C for 4 hours, followed by overnight ligation at 16 °C. Crosslinks were reversed by incubation at 65 °C overnight, and the Low Melting Point Agarose was digested with 0.4 U Agarase for 1 hour at 42 °C. The purified DNA was fragmented with AluI for 1 hour at 37 °C. Next-generation sequencing (NGS) libraries were prepared using the KAPA HyperPrep kit with modifications. The volumes for the end repair, adapter ligation, and PCR reactions were reduced by 5-fold, 5-fold, and 2-fold, respectively, and adapters were diluted to 300 nM. A biotin pull-down was performed with Dynabeads MyOne Streptavidin C1 beads after the adapter ligation step, followed by 25 cycles of PCR. Genomic libraries were sequences using BGI DNBseq G400 machine in 2x150 paired-end mode.
identifiers.namespace: GEO
identifiers.id: GSM8767148_r1