GSM3580955: Four-cell replicate 1 nuclear transfer; Bos taurus; RNA-Seq
Pools of 10 oocytes or embryos were chosen after visual inspection for each biological replicate to minimize variance caused by a single specimen. IVF and SCNT embryos were cultured in vitro under identical conditions (protocol IVF1; (Hiendleder S, et al. (2006) Tissue-specific effects of in vitro fertilization procedures on genomic cytosine methylation levels in overgrown and normal sized bovine fetuses. Biology of reproduction 75(1):17-23.)) to the blastocyst stage (day 7). The following cell stages were collected: GV and MII oocytes (one set used as reference for both IVF and SCNT embryos) and embryos at the four-cell, eight-cell, 16-cell, and blastocyst stage (both embryo types). Pools of 10 oocytes or embryos per stage were frozen and stored at ‑80 °C until analysis. Frozen pools of 10 oocytes or embryos were thawed and lysed in 10 µL of lysis buffer (Prelude kit, NuGEN). The Ovation RNAseq v2 kit (NuGEN) was used for cDNA generation and single primer isothermal amplification (SPIA). The amplified cDNA was bead-purified (AmpureXP, Beckman-Coulter) and fragmented by sonication (Bioruptor, Diagenode; 25 cycles 30 seconds on/30 seconds off), and Illumina-compatible sequencing libraries were constructed from 500 ng of fragmented cDNA by ligation of adapters with sample-specific barcodes. The libraries were amplified (KAPA hifi polymerase, eight cycles, 95 °C 80 seconds, 55 °C 30 seconds, 72 °C 60 seconds) and quantified on a Bioanalyzer 2100 (Agilent) before being pooled at 10 nM concentration for multiplexed sequencing. Three biological replicates of each stage were sequenced on an Illumina GAIIx (single-read, mean coverage of 20 × 10e6 reads, 80-base read length).