title: Illumina HiSeq 2000 sequencing; RNA-seq of estrogen (E2) induced changes of mRNA expression in male and female blastocysts.
design: RNA-seq of estrogen (E2) induced changes of mRNA expression in male and female blastocysts.
library_name: Sample 22_s
library_construction_protocol: We used German Landrace sows, which were inseminated with Pietrain sperm. At day 10 of pregnancy, the uterus was flushed with 10 ml and another 50 ml of phosphate-buffered saline (autoclaved, ph 7.4). All embryos were transferred into a petri-dish containing PBS. Single embryos were shock frozen in liquid nitrogen and stored at -80 °C. A low dose close to the no-observed effect level (NOEL) and a high dose (10 and 1000 µg E2/kg body weight daily, respectively) were orally applied to sows from insemination until sampling at day 10 of pregnancy. They were compared to carrier-treated controls.  Total RNA from single embryos was extracted using the AllPrep RNA/DNA Micro Kit (Qiagen, Hilden, Germany) following the manufacturers protocol for cells with slight modifications. In brief, 700 µl of Buffer RLT Plus supplemented with 1% β-mercaptoethanol was added to the frozen embryo. Disruption was achieved by pipetting up and down and by a single brief vortexing. Homogenization was performed using a syringe and needle. After centrifugation of the lysate using a DNA spin column, the flow-through was processed following the protocol for “purification of total RNA containing small RNAs from cells”. In order to improve RNA purity, the column was incubated with Buffer RPE at step D3 and D4 before centrifugation for 4 min and 2 min, respectively. RNA elution was repeated using the first eluate to increase the final concentration.  Library preparation was performed using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc, California, USA) according to the manufacturer's protocol.
attributes.tag: Experimental Factor: compound
attributes.value: 17beta-estradiol
attributes.tag: Experimental Factor: dose
attributes.value: 10