title: GSM1466810: T-WGBS for rhesus monkey sperm; Macaca mulatta; Bisulfite-Seq
library_construction_protocol: Cumulus–oocyte complexes were collected from animals by laparoscopic follicular aspiration 32–35 hrs following rhCG administration. Follicular contents were placed in Hepes-buffered Tyrode's albumin lactate pyruvate (TALP) medium containing 0.3% BSA at 37°C. Cumulus-oocyte complexes were exposed to hyaluronidase (0.5 mg/mL, <1 min) in TALP-Hepes to strip of cumulus cells. Oocytes that were mature (MII) at collection were placed in chemically defined, protein-free hamster embryo culture medium-10 (HECM-10) at 37 °C in 5% CO2 until inseminated (within 24 hrs) with capacitated, hyperactivated spermatozoa diluted to a final concentration of 2 × 105 /mL in 50-μL drops of TALP for fertilization. After co-incubation of oocytes and spermatozoa for 12–16 h, oocytes were examined for the presence of two pronuclei and two polar bodies as evidence of fertilization. Then the polar bodies were mechanically dissected using Piezo-Micromanipulator to eliminate the DNA contamination of polar bodies and fertilized ova were washed to remove sperms and then cultured in HECM-10 containing 10% FCS (HyClone Laboratories Inc.) to allow embryo development. Culture medium was replaced every other day. The embryos were collected at constant time point (zygotes,10-20 hrs post fertilization(hpf); 2-cell embryos, 24-30 hpf; 8-cell embryos, 2 days post after fertilization (dpf); morula, 4-5 dpf; ICM, 6-7 dpf. Zona pellucidae of embryos were removed by brief exposure (45-60 s) to 0.5% Pronase in culture medium. For ICMs collection, zona-free blastocysts were exposed to rabbit anti-rhesus spleen serum (Axell Labs, Westbury, NY, USA) (1:5) for 30 min at 37 °C. After extensive washing in ESC derivation medium, embryos were incubated in guinea pig complement (Sigma) (1:5) for an additional 30 min at 37 °C and lysised trophectoderm cells were removed from ICMs by pipetting through a fine pulled glass needle. All samples were carefully washed in PBS-EDTA to remove any contaminants. Total 25 oocytes donor monkeys were used in this study and exception of ICM, all the sperms were derived from same male monkey. Transposome complexes were first generated by incubating 2.5 µL of 10 µM annealed adaptors with 2.5 µl 100% glycerol and 5 µl Ez-Tn5 transposase (Epicentre) for 30 min at 25°C. Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen) and purified by AMPure XP magnetic beads. The purified DNA (~0.5 ng, spiked with 10pg of unmethylated lambda DNA) was then incubated with 4 µL Nextera HMW Buffer (Epicentre-Illumina), nuclease-free water (Ambion) to 16 µL and 4 µL prepared Tn5mC transposomes for 12 min at 55°C followed by purification using 36 µl (1.8X) Agencourt AMPure XP magnetic beads and the DNA was eluted in 14 µL EB buffer. An extension step was then performed by adding 2 µL of 10X Thermopol reaction buffer (NEB), 2 µL 10mM 5-Methylcytosine dNTP Mix (Zymo Research, D1030), 1 µl of Bst DNA Polymerase Large Fragment (NEB, M0275s) to each reaction and incubating for 20 min at 65°C. Each reaction was spiked with 200 ng of sonicated unmethylated lambda DNA (200-400 bp) and then subjected to bisulfite conversion using a Zymo EZ DNA Methylation Kit (D5100) according to recommended protocols with a 14h 50°C dark incubation and 22 µL (2 x11 µL) elution. The purified DNAs were then amplified using 25 µL Kapa 2G Robust Hot Start Ready Mix (Kapa Biosystems), 1 µL 50X Nextera Primer Cocktail (Illumina - compatible) and 1 µL barcoded Illumina-compatible Adaptor 2 (8-bp index) on the thermocycler with the following parameters: 1 min at 95°C; (20 sec at 95°C; 30 sec at 60°C; 45 sec at 72°C) X (10-15) cycles. The prepared libraries were analyzed by Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by qPCR and then used for cluster generation and pair-end sequencing with 90 bp reads (PE90) on Illumina Hiseq 2000.