Instrument: Illumina Genome Analyzer IIx
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Frozen pools of ten oocytes or embryos were thawed and lysed in 10 µl of Lysis Buffer (Prelude kit from NuGEN, San Carlos USA). cDNA was generated and amplified with the ovation RNAseq v2 kit (NuGEN, San Carlos, USA). In brief 1 µl of the lysate was used for mixed random-/polyA-primed first-strand cDNA synthesis. After second strand synthesis the double-stranded cDNA was amplified by single primer isothermal amplification and the amplified cDNA was bead-purified (AmpureXP, Beckman-Coulter, Brea, USA) and fragmented by sonication (Bioruptor, Diagenode, Liege Belgium; 25 cycles 30 sec on/30 sec off). 500 ng of fragmented cDNA were used for preparation of Illumina-compatible sequencing libraries using the NuGEN Rapid library kit according to the manufacturer’s protocol. Adapter ligation was done with sample-specific barcodes. The resulting library was amplified (KAPA hifi polymerase, 8 cycles, 95°C 80 sec, 55°C 30 sec, 72°C 60 sec) and quantified on a Bioanalyzer 2100 (Agilent, Santa Clara, USA). Barcoded libraries were pooled at 10 nM concentration for multiplexed sequencing. Three replicates of each stage were sequenced on an Illumina GAIIx to a mean coverage of 20 Mio reads each. Sequencing runs were done in single-read mode with an 80 base read-length.