title: GSM8767331: Chicken oocyte LBC stage nucleus, poly-A, strand specific, single nucleus, biol rep 1; Gallus gallus; RNA-Seq
library_name: GSM8767331
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 frozen immediately. Total RNA was isolated from the nuclei using TRIzol reagent (ThermoFisher Scientific), according to the manufacturer's recommendations. The RNA samples were sent to BGI company to generate transcriptomic libraries using experimental workflow including the following steps: 1) oligoT-enrichment; 2) RNA fragmentation; 3) first-strand synthesis with random primers; 4) second strand synthesis with dUTP instead of dTTP; 5) end-repair, 3'-adenylation and adaptors ligation; 6) Uracil-DNA-Glycosylase treatment followed by PCR amplification and circularization; 7) Sequencing on DNBSEQ in PE100 paired-end mode.
identifiers.namespace: GEO
identifiers.id: GSM8767331_r1