Rnaout kit

Total RNA was extracted from the different tissues of litchi and tobacco using the RNA_OUT kit (Tiandz, Beijing, China). DNase I (TaKaRa, Japan) was added to remove genomic DNA, and RNase-free columns (Tiandz,) were used to purify the total RNA. Then, cDNA was synthesized from total RNA (2 μg) using oligo (dT) primers according to the manufacturer's instructions of PrimeScript™ RT-PCR Kit (TaKaRa).

Total RNA was extracted from peels according to the protocol of the RNAout kit (Tiandz, Beijing, China) and genomic DNA was removed by DNase I (TaKaRa, Dalian, China). RNA quality was analyzed by 1.0% agarose gel and its concentration was quantified by a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA integrity number (RIN) values (>7.0) were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Construction of RNA-Seq libraries was performed by the Biomarker Biotechnology Corporation (Beijing, China). mRNA was enriched and purified with oligo (dT)-rich magnetic beads and then broken into short fragments. The cleaved RNA fragments were reversely transcribed to the first-strand cDNA using random hexamer primers. The second-strand cDNA was synthesized using RNase H and DNA polymerase I. The cDNA fragments were purified, end blunted, ‘A’ tailed, and adaptor ligated. The distribution sizes of the cDNA in the three libraries were monitored using an Agilent 2100 Bioanalyzer. Finally, the three libraries were sequenced using an Illumina HiSeq™ 2500 platform.

Total RNA was extracted from the samples using an RNAout kit (Tiandz, Beijing, China), and cDNA was synthesized with a RevertAid First Strand cDNA Synthesis kit (MBI, USA). Based on the flowering tartary buckwheat transcriptome database constructed by our laboratory (data not shown), a novel bHLH gene (designated as FtbHLH3) was isolated using specific primers. The gene sequence was analyzed via the NCBI database. Multiple amino acid sequence alignments were performed using ClustalX, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method. The primers are listed in Supplementary Table S1.

Total RNA was extracted from hyphae or banana roots using an RNA out kit (Tiandz, Beijing, China), following the manufacturer’s instructions. Genomic DNA was eliminated by RNase-free Recombinant DNase I (Takara, Kusatsu, Japan). The first-strand cDNA was synthesized by AMV Reverse Transcriptase (Takara, Japan) and quantitative real-time reverse transcriptase PCR (RT-PCR) was carried out in a StepOne real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) with the Fast SYBR Green Master Mix (Applied Biosystems). FocEF1α was used as the endogenous reference gene and the relative expression of each gene under different conditions was determined using the 2^-∆∆CT method. Expression of all the samples was examined in three biological replications with two technical duplicates each. All the primer pairs used are listed in Table S2.

Total RNA from different litchi organs/tissues was extracted using the RNA_OUT kit (Tiandz, Beijing, China), and cDNA was synthesized from total RNA (2 μg) using oligo dT primers and M-MLV reverse transcriptase, according to the manufacturer’s instructions (Invitrogen, USA), in a total volume of 20 μL. LcCWIN transcript levels were measured using qRT-PCR, as previously described^{37 (link)}. The specific real-time PCR primers are listed in Table S2. We analyzed the expression in biological triplicate samples. Real-time PCR reactions were normalized to Ct values for litchi LcActin (HQ615689) and LcGAPDH (JF759907). The relative expression levels of the target genes were calculated using the 2^△△Ct method^{38 (link)}.

Total RNA samples were extracted using an RNA Out Kit (Tiandz, Beijing, China) following the manufacturer's instructions. For each sample, 1 μg of RNA was reverse‐transcribed into cDNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara). The qRT‐PCR mixtures (each contained 10 μl of 2 × SYBR Premix Ex Taq buffer, 1 μg of synthesized cDNA and 10 μmol of each gene‐specific primer in a final volume of 20 μl) were prepared using the SYBR Premix Ex Taq kit (Takara). The qRT‐PCR was performed with three technical replicates in a CFX96 real‐time PCR system (Bio‐Rad). FocEF1α (Foc) and MusaActin (banana) were used as internal controls to normalize the data. The relative expressions of genes were calculated using the 2^−ΔΔCT method. The gene‐specific primers used in the qRT‐PCR analysis are listed in Supporting Information Table S1. The abbreviations of genes are shown in the Table S2.

The ovaries and testes were dissected from 3^rd day, 6^th day of fifth instar larvae and 2^nd day of pupae. Total RNAs were isolated from 20 ovaries of silkworm^+Bmovo-1 and WT silkworms, respectively, at the 3^rd day, 6^th day of fifth instar larvae and 2^nd day of pupae using RNAout Kit (Tiandz, Mianyan, China), followed by treatment with RNase-free DNaseI to remove possible contamination with genomic DNA according to the protocol. RNA quality was verified using agarose gel electrophoresis and NanoDrop ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE, USA).