454 sequencing of porcine expanded & hatched blastocysts
RNA was first extracted using the PicoPure RNA kit (Arcturus) from 20 expanded (XB) and 5 hatched (HB) blastocysts independently. High quality total RNA was obtained after DNase treatment using an RNase-Free DNase kit according to the protocol from Qiagen. The Bioanalyzer RNA 6000 Pico LabChip (Agilent Technologies) was used to evaluate the total RNA. 61 ng and 91 ng of total RNA were obtained from HB (RIN=8.4) and XB (RIN=8.2) respectively. Ten ng from each RNA sample was used to synthesize two cDNA libraries using the WT-Ovation Pico RNA amplification system and WT-Ovation Exon Module from NuGEN. Finally, the qualities and quantities of the libraries were assayed on the Bioanalyzer RNA 6000 Pico LabChip (Agilent Technologies). Approximately 10 µg & 6 µg of cDNAs were obtained from HB (with fragment sizes ranging from 133-8000 bp) and XB (with fragment sizes rangeing from 130-4000 bp) respectively. A total of 16 µg of cDNA was combined from HB & XB. 10 µg of this pooled cDNA was nebulized with the nebulization kit supplied with the GS Titanium Library Preparation kit (Roche/454 Life Sciences, CT). Sequencing runs were done using a high-throughput pyrosequencing Genome Sequencer FLX (454 Life Sciences) at the Interdisciplinary Center for Biotechnology Research (University of Florida).