title: GSM1929617: in_vivo_male_A4; Bos taurus; RNA-Seq
library_construction_protocol: The developmental stage and the quality of the blastocysts, according to IETS standards, was determined for all the embryos by the same three trained individuals. Only early blastocysts of quality 1 were selected for the study. Selected blastocysts were washed three times in RNase free PBS (Phosphate buffered saline; Ambion), placed individually in 2 µL of lysis buffer consisting in 5mM DTT (DT-Dithiothreitol; Promega), 4U/ µL RNasin Plus RNase inhibitor (Promega) and 0.64 µM Igepal (Sigma) in RNase free water (Sigma) and immediately stored on -80°C. RNA was extracted using the RNeasy micro kit (Qiagen) excluding RNase treatment. Manufacturer´s instructions were followed with a single modification performed in the elution step, when 14 µL of RNase free water was passed through the column two times. Concentration and quality of the total RNA extracted were examined using Quant-iT RiboGreen RNA Assay kit (Life Technologies) and RNA 6000 Pico Chip (Agilent Technologies), respectively. Subsequently, 1 ng of RNA was used to start the library preparation. First, cDNA was synthesized using the 'SMARTer Ultra low input RNA for Illumina, High Volume kit' (Clontech) mainly following to manufacturer's instructions. For the PCR reaction 12 cycles were chosen. Second, the 'Low input library prep kit' (Clontech) was used to prepare the libraries for sequencing. Libraries were prepared according to manufacturer's instructions, 4+6 cycles were chosen during the PCR reaction in the protocol. Libraries were quantified by qPCR, according to Illumina's protocol 'Sequencing Library qPCR Quantification protocol guide', version February 2011. A high sensitivity DNA chip (Agilent Technologies) was used to control the size, distribution and quality of the libraries. Sequencing was performed by a rapid run on 2 lanes of the Illumina Hiseq-2000 sequencer using 2x100 bp paired-end reads; twelve samples were run per lane in equimolar quantities.
attributes.tag: GEO Accession
attributes.value: GSM1929617