title: GSM3475608: GV OPU; Bos taurus; Bisulfite-Seq
library_construction_protocol: Sample collection: Pools (n = 10) of germinal vesicles (GV) collected at the abattoir (Vigneault et al. 2009), GV collected by OPU (Landry et al. 2016) and 7-day blastocysts produced in vitro (Nivet et al. 2012) starting from abattoir or OPU GV were used in the experiment. Associated granulosa cells were obtained from 113-month-old (aging) or 6-month-old (young) cows (Landry et al. 2016). The mitochondrial abundancy in oocytes and preimplantation embryos (>200,000 mtDNA copies) does not require mitochondrial enrichment prior to mtDNA purification. For granulosa cells, an enrichment procedure must be use where mitochondria are isolated with hypotonic buffer followed by differential centrifugation.  Briefly cells were suspended in 500 µL of RSB (1X) hypotonic buffer in a 1.0 mL glass homogenizer tube using 10 strokes in a B-pestle, diluted with 350 µL of 2.5X MS buffer and centrifuged at 1,300 x g for 5 min at 4°C. The pellet was resuspended and washed again (1,300 x g for 5 min at 4°C) with 500 µL of 1X MS buffer and the two supernatants were pooled in a clean tube and centrifuged at 16,000 xg for 15 min at 4°C. The pellet was re-suspended in 500 µL of 1X MS buffer and washed (16,000 xg for 15 min at 4°C). Mitochondrial DNA was extracted from the final pellet using ZR Genomic DNA – Tissue MicroPrep (Zymo Research) and suspended in 40 µL of DNA elution buffer, digested with plasmid-safe ATP-dependent DNase (Epicenter) in a final volume of 50 µL for 2 h at 37°C followed by 30 min at 70°C then purified using ZR Genomic DNA – Tissue MicroPrep without proteinase K digestion. The purified DNA was suspended in 10 µL of elution buffer. Pools of 10 GV or blastocysts were digested with proteinase K for 20 minutes at 50°C in M-digestion buffer (1X) to obtain a final volume of 20 µL. Granulosa cell mtDNA was mixed with 10 µL of 2X M-digestion buffer and brought to a final volume of 20 µL using the reagents from the EZ DNA Methylation-Direct kit (Zymo Research). Since it has been reported that accessibility of human mtDNA to bisulphite varies depending on the tissue of origin (saliva or blood) and can affect methylation pattern analysis (Liu et al., 2016), a restriction enzyme was used (200 U of Sal1 for 4 h at 37°C) to linearize the bovine mtDNA samples prior to treatment with 130 µL of Lightning Conversion Reagent from the Pico Methyl-Seq Library Prep kit (Zymo Research). The WGBS library protocol was followed as recommended by the manufacturer for 10 pg to 1 ng of DNA with 10 cycles for the final two PCR amplification rounds. Purified libraries were suspended in 12 µL of DNA elution buffer.
attributes.tag: GEO Accession
attributes.value: GSM3475608