CS4H-3
The total RNA was extracted from the collected locust brains by the TRIzol reagent (Invitrogen) according to the manufacturers protocol. The quantity and purity of total RNA extracted as described above were determined with RIN value >7.0 by ND-1000 Nanodrop (Thermo Fisher, USA) and Agilent 2200 Tapestation (Agilent Technologies, USA). The RNA integrity was examined by agarose gel electrophoresis. Then ribosomal RNA was removed from total RNA using EpicentreRibo-Zero rRNA Removal Kit, (Illumina company, USA). The rRNA depleted RNA was put to perform randomly fragmented into short segments and instantly used as template to synthesis the first cDNA by random hexamers. Next, the second cDNA was synthesized and RNA was digested by RNase H. The double-stranded DNA then was purified and added with bases A and adaptor in the 3-end. After selection by AMPureXP beads, the second strand cDNA containing U was degraded by enzyme USEB. Finally, the cDNA library for sequencing was constructed by PCR amplification. Then the quality of cDNA library was also inspected and the sequencing was performed on an Illumina Hiseq3000.