Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: All cumulus cells were removed by repeatedly pipetting oocytes with a narrow-gauge pipette that was just slightly larger than the oocytes while observing oocytes with a stereomicroscope. Using the PicoPureTM RNA Extraction kit, total RNA was isolated from each sample following the manufacturer protocol, including a DNAse digestion (RNase-Free DNase Set; Qiagen, Hilden, Germany) to remove any contaminating DNA. SPIA libraries: 100 ng of each RNA sample was processed with the Ovation RNA-Seq System v2 using Ribo-SPIATM Technology (NuGen, San Carlos, CA). After this initial processing, a Covaris-2 sonicator was used to mechanically fragment the cDNA to an average of 300 bp. This was then followed by a brief S1 nuclease digestion as previously described (Head et al., 2011). After bead purification, the cDNA was processed through the Ovation Ultralow DR Multiplex Systems 1-16 (NuGen) for end repair, adaptor ligation and final library amplification. SoLo libraries: cDNA libraries were produced with Nugen Ovation SoLo RNA-Seq(NuGEN, San Carlos, CA). After bead purification, the cDNA was processed: end repair, adaptor ligation and first round library amplification and purification. Then 20 ng of each library was used for the remainder of library preparation, which included use of InDA-C primers for rRNA depletion, as well as 2nd round library amplification and purification steps. SoLo Custom R1 primer was used instead of a standard Illumina R1 sequencing primer for the sequencing of the libraries.