GSM4173949: RNA-seq WT repl4; Drosophila melanogaster; RNA-Seq
The Trizol solution was defrosted on ice filled up to 500 µl and incubated for 5 min at room temperature.  After the addition of 100 µl chloroform and vigorous mixing, the samples were spun in a table top centrifuge at 4 °C, 12.000 g for 15 min in order to separate the organic and aqueous phase. The aqueous phase was transferred in to 300 µl of chloroform and the step the first step was repeated. Subsequently the aqueous phase was transferred into isopropanol containing 20 µg of glycogen. The RNA was precipitated at -20 °C at least overnight and pelleted by centrifugation (4 °C, 12.000 g for 60 min). The pellet was washed with 80% ethanol and resuspended in DNase-digestion buffer (2 U TurboDNase Ambion, 1x TurboDNase buffer Ambion). After digestion for 30 min at 37 °C the reaction was stopped by adding EDTA to a final concentration of 15 mM and heating the samples to 75 °C for 10 min. The RNA was quantified using Qubit RNA BR Assay kit (ThermoScientific Q10210) and integrity was assessed on agarose gels or the fragmentanalyzer (Agilent). The Illumina TruSeq Stranded Total RNA protocol was used for library preparation for next generation sequencing.
GEO
GSM4173949
GEO Accession
GSM4173949