title: GSM5664531: WSD_3; Macaca mulatta; RNA-Seq
library_construction_protocol: COS protocols were performed. Briefly, recombinant human gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), were administered to promote the development of multiple ovarian follicles. Female rhesus macaques were anesthetized for laparoscopic follicular aspirations 36 hours (hr) after a bolus of human chorionic gonadotropin (hCG) was administered to induce events necessary for the re-initiation of meiosis. Two individual follicle aspirates per ovary (N=4 each animal) were collected manually using a low dead space 3ml syringe with a 22-gauge X 1.5inch needle (Ulticare, UltaMed Inc., Excelsior, MN). Individual follicle aspirates were centrifuged to separate the follicular fluid (FF) from the cumulus oocyte complexes (COCs) and the granulosa cells (GCs). FF was stored at -80°C until analysis and the oocytes from the individual aspirates were denuded from the surrounding cumulus cells (CCs) of the COCs by gentle micropipetting. The remaining follicles were aspirated in bulk using a (Ulticare, UltaMed Inc., Excelsior, MN) connected to a vacuum system. Bulk aspirated COCs were collected in Tyrode's albumin lactate in pyruvate (TALP)-HEPES medium with 0.3% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO) and 1% Heparin sodium salt solution at 37°C. Oocytes from the bulk aspirates were denuded by gentle micropipeting in TALP-HEPES medium containing 0.3% BSA and 3% hyaluronidase (Sigma-Aldrich). The developmental stage of each oocyte was recorded and categorized as either immature germinal vesicle (GV), maturing metaphase I (MI), mature metaphase MII (MII), or degenerated. Individual oocytes were placed in pre-equilibrated 100 μL TALP complete drops in a 10-well IVF dish (LifeGlobal, Guildford, CT) in 5% CO2 at 37°C until IVF. Blastocysts were collected from all 4 treatment groups, the zona pellucida removed, washed with Ca2+ and Mg2+-free PBS, and frozen at -80°C until RNA isolation. To analyze the effect of the treatments rather than embryo morphology, 38 blastocysts (N=12 C, N=12 T, N=10 WSD, and N=4 T+WSD) that did not exhibit cellular fragmentation or asymmetrical/multipolar divisions were chosen for RNA-seq analysis. RNA was extracted from the blastocysts using the ARCTURUS PicoPure RNA Isolation Kit (ThermoFisher Scientific KIT0204, Waltham, MA), cDNA prepared using the SMART-Seq v4 Ultra Low Input RNA Kit for sequencing (TakaraBio, Shiga, Japan) and the amplified cDNA purified using the Agencourt AMPure XP Kit (Beckman Coulter, Brea, CA), all according to the manufacturer's instructions. cDNA was then sheared to approximately 250 base pairs in length using a Covaris M220 sonicator.  Sheared cDNA was resuspended in Tru-Seq Resuspension Buffer and libraries prepared using a Tru-Seq Nano kit (Illumina, San Diego, CA) according to the manufacturer's instructions, except that 16 cycles of amplification were performed to account for low input samples. Fragment size was measured using a Fragment Analyzer 5200 and samples quantified with quantitative PCR and pooled at equimolar concentration. Multiplexed samples were sequenced across seven lanes of a single-read, 75 cycle run on an Illumina HiSeq 4000 sequencer.