Tb green premix ex taq tli rnaseh plus

For transcriptomic analysis, 3’ RNA-seq analysis was performed as previously described [49 (link)]. Briefly, the guts of w^Dah flies were dissected. Crop and Malpighian tubules were carefully removed. Triplicate samples were prepared for each experimental group. Total RNA was purified using a ReliaPrep RNA Tissue Miniprep kit (Promega, z6112). The RNA was sent to Kazusa Genome Technologies to perform the library preparation and sequencing. Raw reads were analysed by the BlueBee Platform (LEXOGEN), which performs trimming, alignment to the Drosophila genome, and counting of the reads. The count data were statistically analysed by the Wald test using DESeq2. The result has been deposited in DDBJ under the accession number DRA015054.
For quantitative RT–PCR analysis, total RNA was purified from 3–4 tissues of female flies using a ReliaPrep RNA Tissue Miniprep kit (Promega, z6112). Quantitative RT-PCR was performed using a OneTaq RT–PCR kit (Promega, M0482S) with qTOWER3 (Analytik Jena), or PrimeScript RT reagent Kit (Takara, RR037A) and TB Green Premix Ex Taq (Tli RNaseH Plus) (Takara, RR820W) with Quantstudio6 Flex Real Time PCR system (ThermoFisher). The primer sequences are listed in Table 2.

Total RNA was isolated with RNAiso Plus Reagents (Takara, Dalian, Liaoning, China) according to the manufacturer’s instructions. cDNA was reverse-transcribed from 800 ng of total RNA using the PrimeScrip RT reagent Kit with gDNA Eraser (Takara). Gene-specific primers with appropriate amplification efficiency (0.95 to 1.05) were screened by a cDNA dilution series (S4 Table). Quantification of gene expression level was performed with TB Green Premix Ex Taq Tli RNase H Plus (Takara) on a LightCycler 480 system (Roche, Basle, Switzerland). Target genes expression was normalized by the commonly used housekeeping gene actin5c (GenBank: AJ862721.1) and calculated using the 2^−ΔΔCt method. Each treatment contained 4 biological replicates and technical triplicates.

Total RNA was extracted using TRIzol™ Reagent (Invitrogen, 15596018) following the general experimental protocol for RNA isolation. Complementary DNA was synthesized from 500 ng of total RNA using PrimeScript™ RT Master Mix (Perfect Real Time) (Takara, RR036A). Quantitative real-time PCR (qRT-PCR) was performed using TB Green^® Premix Ex Taq™ (Tli RNaseH Plus) (Takara Bio, Mountain View, CA, USA, RR420A) and the LightCycler^® 480 Instrument II (Roche). The mRNA transcript abundance was normalized to that of Actb and Hprt. The qRT-PCR primer sequences are described in Table S1.

The cDNA was synthesized via the PrimeScript^TM RT reagent kit (TaKaRa, Dalian, China) according to instructions from the manufacturer. An RT-qPCR was carried out in a CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA) with TB Green ^® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa, Dalian, China). The B. cinerea actin gene BcactA (Bcin16g02020) was used as internal control. The relative expression of each gene was evaluated using the ^ΔΔCT method [22 (link)]. All primers used for the RT-qPCR analyses are listed in Table S1. The RT-qPCR assay was repeated three times, each with three biological replicates.

Total RNA was extracted using Trizol reagent, and the 1^st strand cDNA was synthesized using Super script II reverse transcriptase, according to the manufactures instruction. qRT-PCR was performed using TB Green Premix Ex Taq (Tli RNase H Plus, Takara Bio, Shiga, Japan) and Applied Biosystems^® StepOnePlus™ real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) as described previously (15 (link)). The primers used for the assay (No. 5-10; 17-40) are listed in Table S1. The specificity of PCR was confirmed by denaturation curve analysis, and PCR products were analyzed by electrophoresis to determine the single amplicon.

Total RNA was extracted with TRIzol (RNAiso Plus) (Takara, Japan). RNA was reversed transcribed into cDNA using the twostep method with PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan). Genomic DNA of miRNA was removed using Recombinant DNase I (RNase-free) (Takara, Japan). miRNA was reversed transcribed into cDNA using the Mir-X® miRNA FirstStrand Synthesis and SYBR® qRT-PCR (Takara, Japan). mRNA qRT-PCR was performed with the TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Japan). miRNA qRT-PCR was performed with Mir-X™ miRNA FirstStrand Synthesis and SYBR® qRT-PCR (Takara, Japan). The primers used are shown in Table 1.

All in vitro infection experiments were performed with a multiplicity of infection (MOI) of 0.1. In vitro samples were collected at 24, 48, and 72 h postinfection, cells were subsequently lysed with RLT buffer, and RNA was extracted using a Qiagen RNeasy kit (Qiagen, Valencia, CA, USA). cDNA was reverse transcribed from RNA extracts (500 ng) using a PrimeScript one-step reverse transcriptase PCR (RT-PCR) kit version 2 (TaKaRa, Japan). qPCR was carried out using TB Green premix Ex Taq (Tli RNase H Plus) (TaKaRa, Japan) with the primers listed in Table 3. Fold changes of target genes in infected samples were calculated by using the threshold cycle (ΔΔC_T) method and normalized to a GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA level (93 (link)). Cell-free supernatants were frozen and thawed before quantification of CXCL11 and IL36G was performed using a human CXCL11 enzyme-linked immunosorbent assay (ELISA) kit (Abcam) and human IL36G ELISA kit (Signalway Antibody), respectively. Statistics were performed with GraphPad Prism 8 software using unpaired t test. P values of <0.05 (*), <0.01 (**), and <0.001 (***) were considered significant. Bars in the figure panels show means and standard errors of the means (SEMs).