title: GSM8767172: Chicken oocyte LBC stage nucleus, NOME-seq, single nucleus, biol rep 11; Gallus gallus; OTHER
library_name: GSM8767172
library_construction_protocol: Chicken oocytes, with diameters ranging from 1 to 2 mm as lampbrush chromosome stage, 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, 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. Individual nuclei were transferred immediately to 3 μL of GC Reaction Buffer (NEB) with 0.2% Igepal for 20 minutes at RT. Reaction of methylation was performed in 1x GC Reaction Buffer supplemented with 160 µM S-adenosylmethionine with 6U GpC Methyltransferase at 37 °C 1 hour. Proteines were digested by proteinase K at 65 °C for 2 hours. For control experiments we also supplemented chicken oocytes nuclei PICO19 and PICO22 with human K562 cells. Libraries for whole genome bisulfite sequencing were prepared using Pico Methyl-Seq Library Prep Kit (Zymo Research) according to the user manual.
identifiers.namespace: GEO
identifiers.id: GSM8767172_r1