title: GSM8651479: CG; Oryctolagus cuniculus; RNA-Seq
library_construction_protocol: A total of 150 female IRA rabbits (35 days old) were randomly divided into three groups, and kept in environmental control rooms for four weeks. The rabbits in the control group (CG) were kept under an NH3 concentration of < 3 ppm. The two treatment groups were kept under NH3 concentrations of 10 ppm (low ammonia concentration, LAC) and 30 ppm (high ammonia concentration, HAC). The oocyte complexes cumulus of the ovarian were taken after slaughter and frozen in liquid nitrogen for examination. Approximately 5ug of total RNA was used to deplete ribosomal RNA according to the manuscript of the Ribo-Zero Gold rRNA Removal Kit (Illumina, cat.MRZG12324, San Diego, USA). After removing ribosomal RNAs, the left RNAs were fragmented (NEBNext® Magnesium RNA Fragmentation Module, cat.E6150S, USA) into short fragments using divalent cations under high temperature. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H(NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection (300-600bp) was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min;8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec;and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300±50bp. At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000following the vendor's recommended protocol.