18°C #1 (replicate b)
Total RNA was extracted from pooled ovaries with TRI Reagent (Ambion) according to the manufacturer’s instructions. rRNA was removed from total RNA extracts with Illumina’s Ribo-Zero rRNA Removal Kit according to the manufacturer’s instructions. rRNA-depleted RNA was then applied to a 10% urea-based denaturing polyacrylamide gel and run for 20 minutes at 1300 V and 60 W along with GeneRuler Ultra Low Range DNA Ladder (Thermo Scientific). The small RNA fraction ranging from approximately 15 nt to 40 nt was excised from the gel and resolved in 30 μl nuclease-free water using Ultrafree-MC and Ultra-0.5 3K centrifugal devices (Amicon) according to the manufacturer‘s instructions. RNA concentration and integrity was tested with Agilent’s 2100 Bioanalyzer system using the Agilent RNA 6000 Nano Kit. NanoDrop 2000 system (Thermo Scientific) was used to quantify inorganic ions and test for polycarbonate contamination. Adaptor ligation was performed using Illumina’s TruSeq Small RNA Sample Pre Kit according to the manufacturer’s instructions. RNA with ligated adaptors was subject to reverse transcription using Invitrogen’s Super Script II Kit. The resulting cDNA was PCR-amplified according to the following thermal cycling profile: 30′’ at 98°C, [10′′ at 98°C, 30′′ at 60°C, 15′′ at 72°C] × 11, 10′ at 72°C. DNA concentration of the libraries was tested with Agilent’s 2100 Bioanalyzer system using the Agilent DNA 1000 Kit. Finally, eight libraries (four probes, two replicates each) were sequenced on one lane on an Illumina HiSeq 4000 system (single end, read length=50).