Reliaprep rna tissue miniprep kit

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.

For the CIF2 peptide treatment, 5‐day‐old seedlings grown on half MS were moved to fresh 1/2 MS medium in the absence or presence of 100 nM CIF2 and incubated for 30 or 120 min. Otherwise, the plants were grown with mesh. Only root parts (around 100 mg) were collected at each time point, and total RNA was extracted using a TRIzol‐adapted ReliaPrep RNA Tissue Miniprep Kit (Promega). Reverse transcription was carried out with PrimeScript RT Master Mix (Takara). All steps were done as indicated in the manufacturer's protocols. The qPCR was performed on an Applied Biosystems QuantStudio3 thermocycler using a MESA BLUE SYBR Green kit (Eurogentech). All transcripts are normalized to Clathrin adaptor complexes medium subunit family protein (AT4G24550) expression. All primer sets are indicated in Table EV3.