biological replicate2 of gland tissue in Chrysomela populi, (injected with dsCpabc35(Cpmrp)-RNA)
Sequence-verified plasmid pIB- Cpabc35 (CpMRP; Gen-Bank accession number KC112554) was used to amplify a 730 bp fragment of cpmrp dsRNA. As a control, a gfp sequence was amplified from pcDNA3.1/CT- GFP-TOPO (Invitrogen). The amplicons were subject to an in vitro-transcription assays according to instructions from the Ambion MEGAscript RNAi kit. The resulting dsRNA was checked by TBE-agarose-electrophoresis and injected into first-instar larvae. Differential expression in the glandular tissue was analysed 10 days after RNAi treatment.  We extracted the total RNA by using the RNAqueous Micro Kit including DNase  treatment(Ambion, Life Technologies) from glandular tissues from three Chrysomela populi larvae.  The RNA integrity was validated by using RNA 6000 Nano labchips on a Bioanalyzer 2100 (Agilent Technologies).  Library preparation for sequencing was carried out with 5 µg of total RNA by using TruSeq™ RNA Sample Prep Kit (illumina). For the RNA sequencing we ran illumina next-generation sequencing technique (RNA-seq), multiplexed two or three samples in one lane.