alias: E-MTAB-6201:Sample 11_s
title: Illumina HiSeq 4000 sequencing; Small RNA-Seq analysis of miRNA isoform (isomiR) expression in single porcine Day 10 blastocysts and analysis of effects of maternal estradiol-17-beta feeding from day of ovulation until Day 10 of pregnancy
design: Small RNA-Seq analysis of miRNA isoform (isomiR) expression in single porcine Day 10 blastocysts and analysis of effects of maternal estradiol-17-beta feeding from day of ovulation until Day 10 of pregnancy
library_construction_protocol: The experimental trial has been previously described (Fürst et al. 2012, Flöter et al, under review). In brief, estrous cycle synchronized German Landrace sows (n=4-6/treatment) were inseminated with sperm of the same single Pietrain boar. Starting from insemination until Day 10, sows were fed with different doses of estradiol-17β (E2; 1, 3, 5(10)-ESTRATRIEN-3, 17β-DIOL, Steraloids, Newport, USA), namely with 10 and 1000 µg E2/kg body weight (bw) twice daily, respectively, or with ethanol carrier only (control group). The E2 concentrations were selected according to reference values for humans (JECFA 2010) and have been reported earlier (Fürst et al. 2012). The low dose corresponds to the 'no observed effect level' (NOEL). One hour after ingestion of the last dosing, sows were slaughtered on day 10 of pregnancy. The uterus was removed and embryos were flushed from the uterus using 10 ml phosphate-buffered saline (PBS, autoclaved, pH 7.4) per horn. All embryos were transferred into a Petri-dish containing PBS, washed twice and then single embryos were snap-frozen in liquid nitrogen and stored at -80 °C. Animals were only included in the RNA sequencing (RNA-Seq) analysis if embryos were at the hatched blastocyst stage. Experiments with sows were performed in accordance with the International Guiding Principles for Biomedical Research Involving Animals, as proposed by the Society for the Study of Reproduction, with the European Convention on Animal Experimentation and with the German Animal Welfare Act. In brief, estrous cycle synchronized German Landrace sows (n=4-6/treatment) were inseminated with sperm of the same single Pietrain boar. Starting from insemination until Day 10, sows were fed with different doses of estradiol-17β (E2; 1, 3, 5(10)-ESTRATRIEN-3, 17β-DIOL, Steraloids, Newport, USA), namely with 10 and 1000 µg E2/kg body weight (bw) twice daily, respectively, or with ethanol carrier only (control group) Total RNA and DNA from single embryos were extracted as reported recently (Flöter et al, under review) 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 embryos. Disruption was achieved by pipetting up and down and by a single brief vortexing. Homogenization was performed using a syringe and needle (20 G). After centrifugation of the lysate using a DNA spin column, the column was stored at 4 °C, while 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. The DNA was purified subsequently. Samples were immediately put on ice. RNA and DNA samples were stored at -80°C and -20°C, respectively. Purity and quantity was assessed spectrophotometrically using the NanoDrop 1000 (peqLab, Erlangen, Germany). Additionally, RNA quantity of embryos was determined using the Qubit (Invitrogen) with the Qubit™ RNA BR Assay. RNA integrity was measured by means of the Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany) with the RNA 6000 Nano Kit (Agilent). The mean RNA Integrity Number (RIN) of the embryo samples was 9.7 ± 0.3 (± SD) NEBNext® Small RNA libraries were prepared starting from 50-100 ng total RNA from individual embryos (n=6 per group, 6 groups in total)
identifiers.namespace: ETH Zurich
identifiers.id: E-MTAB-6201:Sample 11_s