SRX039506 FY9BXRK SRP005484 SRA029132 submitted by University of Alberta on 2011-01-24 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). SRS160758 Day 5-6 porcine blastocysts HB_01 EST TRANSCRIPTOMIC cDNA 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX044479 GHRM6LN SRP005484 SRA029132 submitted by University of Alberta on 2011-01-24 RNA was first extracted using Arcturus® PicoPure® RNA Isolation Kit from 150 pooled embryos and oocytes. High quality total RNA was obtained after DNase treatment using RNase-Free DNase kit according to the protocol from Qiagen. Bioanalyzer RNA 6000 Pico LabChip (Agilent Technologies) was used to evaluate the total RNA. A yield of 25.41ng of total RNA (RIN=8.4) was obtained and used for first-strand cDNA synthesis according to Clontech manual of Super SMART™ PCR cDNA Synthesis Kit with some modifications. Reverse transcription (RT) was carried out with the SMART™ MMLV reverse transcriptase and the RT reaction was extended to 90 minutes at 42°C. Following second strand amplification, 3.1 µg of purified cDNA was obtained using a QIAquick mini elute kit (QiagenValencia, CA). The cDNA library was normalized according to the protocol described in the Trimmer Direct Kit (Evrogen, Russia). In brief, 1 µg of cDNA were incubated at 98°C for 2 minutes followed by incubation at 68°C for 5 hours in the provided hybridization buffer in (50 mM Hepes, pH7.5 and 0.5 M NaCl). The optimal digestion was treated with 1/8 units of duplex specific nuclease (DSN). The normalized cDNA was then amplified from 1 µl of DSN-treated cDNA by PCR reactions (11 cycles) involving: 95°C for 1 minute, followed by 12 cycles with 95°C for 15 seconds, 64°C for 20 seconds and 72°C for 3 minutes, with a final extension of 72°C for five minutes and clean up with a Qiaquick mini elute PCR column. Then 20 µg of normalized cDNA library was obtained and 15 µg of this sample was nebulized with the nebulization kit supplied with the GS Titanium Library Preparation kit (Roche/454 Life Sciences, CT). In order to improve the sequencing yield by reducing the length of long poly(A) tail problem during 454 sequencing, an additional BAL 31 nuclease digestion was carried out on the rest of the cDNAs to remove homopolymers according to the protocol provided by USB Corporation. The reaction was performed at 30°C for 2 minutes and the nuclease activity was stopped by adding 0.5M EGTA. Sequencing runs were done using high-throughput pyrosequencing Genome Sequencer FLX (454 Life Sciences) at McGill University and Genome Quebec Innovation Centre. SRS172986 In vitro generated porcine embryos PIVT or PIVT-BAL EST TRANSCRIPTOMIC cDNA 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX044480 GK5Z48F SRP005484 SRA029132 submitted by University of Alberta on 2011-01-24 RNA was first extracted using Arcturus® PicoPure® RNA Isolation Kit from 121 pooled embryos and oocytes. High quality total RNA was obtained after DNase treatment using RNase-Free DNase kit according to the protocol from Qiagen. Bioanalyzer RNA 6000 Pico LabChip (Agilent Technologies) was used to evaluate the total RNA. A yield of 50 ng of total RNA (RIN=7.7) was obtained and used for first-strand cDNA synthesis according to Clontech manual of Super SMART™ PCR cDNA Synthesis Kit with some modifications. Reverse transcription (RT) was carried out with the SMART™ MMLV reverse transcriptase and the RT reaction was extended to 90 minutes at 42°C. Following second strand amplification, 3 µg of purified cDNA was obtained using a QIAquick mini elute kit (QiagenValencia, CA). The cDNA library was normalized according to the protocol described in the Trimmer Direct Kit (Evrogen, Russia). In brief, 880 ng of cDNA were incubated at 98°C for 2 minutes followed by incubation at 68°C for 5 hours in provided hybridization buffer (50 mM Hepes, pH7.5 and 0.5 M NaCl). The optimal digestion was treated with 1/8 units of duplex specific nuclease (DSN). The normalized cDNA was then amplified from 1 µl of DSN-treated cDNA by PCR reactions (12 cycles) involving: 95°C for 1 minute, followed by 12 cycles with 95°C for 15 seconds, 64°C for 20 seconds and 72°C for 3 minutes, with a final extension of 72°C for five minutes and clean up with a Qiaquick mini elute PCR column. Then 20 µg of normalized cDNA library was obtained and 15 µg of this sample was nebulized with the nebulization kit supplied with the GS Titanium Library Preparation kit (Roche/454 Life Sciences, CT). In order to improve the sequencing yield by reducing the length of long poly(A) tail problem during 454 sequencing, an additional BAL 31 nuclease digestion was carried out on the rest of the cDNAs to remove homopolymers according to the protocol provided by USB Corporation. The reaction was performed at 30°C for 2 minutes and the nuclease activity was stopped by adding 0.5M EGTA. Sequencing runs were done using high-throughput pyrosequencing Genome Sequencer FLX (454 Life Sciences) at McGill University and Genome Quebec Innovation Centre. SRS172987 In vivo generated porcine embryos PIVV or PIVV-BAL EST TRANSCRIPTOMIC cDNA 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium