Dnase 1 kit

Harvested tissue samples were ground to powder in liquid nitrogen. Total RNA was extracted from various tissues using TRIzol reagent (Invitrogen, Carlsbad, USA) following the manufacturer’s instructions. Genomic DNA was removed using a DNase I kit (Qiagen, Hilden, Germany), and RNA quality was detected with an Agilent 2100 bioanalyzer (Agilent Technologies, CA, USA) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, USA).
For qRT–PCR (quantitative real-time PCR) analysis, 1 μg of total RNA was used to synthesize first-strand cDNA using the Prime-Script RT Reagent Kit (Takara, Dalian, China). qRT–PCR was performed on an ABI 7500 Real Time PCR system (Applied Biosystems™, Foster City, USA) using TB Green™ Premix Ex Taq™ II (TaKaRa, Shiga, Japan). Specific primers for the CBL and CIPK genes were designed using the IDT PrimerQuest online tool (https://sg.idtdna.com/PrimerQuest/Home/Index). An actin gene (CiPaw.03G124400) that used in previous studies was applied as an internal control for normalization [37 (link)]. Relative quantification of CBL and CIPK genes was determined by the 2^-∆∆Ct method [38 (link)]. The PCR cycling conditions were as follows: initial denaturation at 95 °C for 30 s, then 40 cycles of 95 °C for 5 s and 60 °C for 15 s.

Total RNAs were isolated from whole bodies of the first- through fifth-instar nymphs, adults and from tissues or the nymph survivors of the dsRNA bioassay with RNeasy mini kit (Qiagen, Germany) according to manufacturer’s instruction. The potential genomic DNA contamination was removed by a treatment with DNase I kit (Qiagen Germany) after the RNA extraction. RNA concentration and quality was determined using a Nanodrop spectrophotometer (Thermo Scientific, USA). The first-strand cDNA were synthesized by reverse transcriptase using ReverTra AceqPCR RT Kit (ToYoBo, Osaka, Japan). The 10-fold diluted first-strand cDNA (2.0μL) was used as template for qRT-PCR.
mRNA abundance of each gene was tested in three technical replicates for each of three biological replicates. qRT-PCR were performed using a qPCR master mix SYBR^® Premix (Toyobo, Osaka, Japan) on ABI 7500 System (Applied Biosystems). The accompanying software was used for qPCR data normalization and quantification. Relative value for the expression level of target gene in development stages and tissues was calculated by the equation Y = 10 ^{(Ct internal- Ct target)/3} x100% [28 ], using the geometric mean of 18S rRNA (JN662398) and β-actin (EU179846) as internal [29 (link)]. Duncan's Multiple Comparison was used to determine differences among tissues and stages. Values of p<0.01 were considered significant.

Total RNA was extracted from approximately 60 mg of tissue for each of the 22 tumor samples using the RNAiso Plus (Takara) and purified using DNase I kit (QIAGEN) based on manufacturer instructions. Quantification of the RNA yields was performed by NanoDrop ND1000 (Thermo Fisher Scientific, Waltham, MA). The quality of the RNA was evaluated by the Agilent 2,100 Bioanalyzer (Agilent, Santa Clara, CA). A cDNA library was prepared according to the protocol of TruSeq RNA Sample Preparation based on manufacturer instructions. The cDNA library of each sample was assessed and validated on Qubit (Qubit ds DNA HS assay kit) and on an Agilent Bioanalyzer 2,100 (HS DNA kit). Further, the cDNA library was normalized and after pooling was then hybridized on a flow-cell v3 (TruSeq E Cluster Kit version 3). Finally, RNA paired-end sequencing was performed on an Illumina HiSeq2000 platform according to the standard protocol using TruSeq SBS Kit (TruSeq SBS Kit version 3—HS). A PhIX control library was used as an in-spike for each line (Huang et al., 2011 (link)). Transcriptome sequencing data are available publicly at the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/) under accession number PRJNA608223.

Total RNA was extracted with RNeasy Micro Kit (QIAGEN), treated with DNase I Kit (QIAGEN), and reverse transcribed using SuperScript III Kit (Invitrogen) as per the manufacturer's instructions.
miRNA qRT-PCR has been described previously (Moltzahn et al., 2011 ). Primers and probes can be found in Supplemental Information.

Overnight cultures of E. coli TolC (OD₆₀₀, ~5) were diluted 1:200 in LB broth and grown to an OD₆₀₀ of 0.1. The culture was subsequently divided into equal parts: one part was treated with 0.25 µg/ml carolacton, and the other part was treated with an equal volume of solvent (methanol). Cells were sampled before treatment and at 5, 15, and 30 min post-addition of carolacton. The samples were transferred to an equal volume of RNAProtect (Qiagen, Germany) and incubated for 5 min at room temperature. Cells were pelleted (13,000 rpm, 2 min), the supernatant was removed, and the pellet was frozen at −80°C. For RNA extraction, the pellets were washed with 0.5 ml nuclease-free water and centrifuged (13,000 rpm, 2 min). RNA extraction was carried out using the miRNeasy minikit (Qiagen, Germany) according to the manufacturer’s instructions for purification of total RNA. The removal of genomic DNA was carried out by the optional on-column DNase I digestion using the DNase I kit (Qiagen, Germany) for 45 min. After the washing steps, the RNA was eluted in 50 µl of nuclease-free water supplied with the kit. To test the integrity of the isolated total RNA and the enriched mRNA, samples were analyzed using the Agilent 2100 Bioanalyzer and the RNA 6000 Pico kit (Agilent, Germany).