Dpse_female_temperature_14h
15 μg Poly(A)+ RNA was selected on oligo-dT25 DynaBeads (Invitrogen), and fragmented at 70°C in fragmentation buffer (2 mM EDTA, 100 mM NaCO3/NaHCO3, pH 9.2). Fragmented RNAs were purified on a 15% TBE-Urea gel (selecting 40-80nt fragments), dephosphorylated by PNK reaction (New England Biolabs), ligated with 3’-adaptor and purified on a 15% TBE-Urea gel. The purified RNAs were phosphorylated by PNK reaction (New England Biolabs), and ligated to 5’ adapter, subjected to reverse-transcription into cDNAs. Prepared cDNAs were amplified by PCR with Phusion High-Fidelity DNA polymerase, purified, and sequenced on Illumina Hiseq platform and single-end reads were generated.