Total RNA was isolated using the TRIzol reagent (Invitrogen; #15596026), treated with DNase I, and purified using RNeasy Mini columns (Qiagen; #74034). The purity and quantity of the total RNA was measured using a Nanodrop 2000 (Thermo Scientific). The integrity of the RNA was assessed using a Bioanalyzer 2000 system (Agilent Technologies).
The library was constructed according to Novogene’s (Beijing, China) procedure, as previously described (13 (link)). Briefly, 3 μg of total RNA were utilized for the RNA sample preparations. rRNA was removed using the Ribo-zero™ rRNA removal kit (Epicentre) and the residual RNAs were cleaned up by ethanol precipitation. Subsequently, sequencing libraries were generated based on the rRNA-depleted RNA by using NEBNext® Ultra™ directional RNA library prep kit from Illumina® (Lincoln, NE) following the manufacturer’s recommendations. After library quality was assessed on an Agilent Bioanalyzer 2100 system, RNA-seq was performed on the Illumina Hiseq 2500 platform by Novogene.
The library was constructed according to Novogene’s (Beijing, China) procedure, as previously described (13 (link)). Briefly, 3 μg of total RNA were utilized for the RNA sample preparations. rRNA was removed using the Ribo-zero™ rRNA removal kit (Epicentre) and the residual RNAs were cleaned up by ethanol precipitation. Subsequently, sequencing libraries were generated based on the rRNA-depleted RNA by using NEBNext® Ultra™ directional RNA library prep kit from Illumina® (Lincoln, NE) following the manufacturer’s recommendations. After library quality was assessed on an Agilent Bioanalyzer 2100 system, RNA-seq was performed on the Illumina Hiseq 2500 platform by Novogene.
Free full text: Click here
