M mlv transcriptase

To verify the presence of putative mycovirus in the strains, cDNAs were synthesized using Moloney murine leukemia virus (M-MLV) transcriptase (Takara Bio, Inc., Japan), and viral sequences were detected by RT-PCR using specific primers designed on the 26 assembled contigs that matched viral sequences (Supplementary Table S2). To complete the sequences of the alphavirus-like virus genomes, the 5′- and 3′-terminal sequences were determined using the SMARTer RACE 5′/3′ kit (Clonetech Laboratories, Mountain View, CA. Primers 5′-1R (5′-ATGGGTTTAAGGAGAGAGTGCGAGAGTCTATGTTCTT GAATGTC-3′) and 5′-2R (5′-CGAGCCAAGAGAATAGAGT AGGAGAG-3′) were used for 5′ random amplification of cDNA ends (RACE) as outer and inner primers, respectively. Primers 3′-1F (5′-AAGCGGCACCTAGACCTACGACATTGGCAGACA-3′) and 3′-2F (5′-GTGACGGAGTACGACCAGTCTT-3′) were used for 3′ RACE as outer and inner primers, respectively.

Total RNAs were extracted from RCC cells after different treatments. Reverse transcription was performed on 2 μg total RNA by M‐MLV transcriptase (Takara, Kusatsu, Japan). After obtaining the cDNA, qPCR was carried out in triplicate using an ABI 7500 Fast instrument (Applied Biosystems, Foster, CA, USA). The primers used are as follows: Sirt5, forward 5′‐GCACAGAGCCTCGCCTT‐3′, reverse 5′‐GTTGTCGACGACGAGCG‐3′; glyceraldehyde‐3 phosphate dehydrogenase, forward 5′‐TCTCTGCTCCTCCTGTTC‐3′, reverse 5′‐GTTGACTCCGACCTTCAC‐3′.

Total RNA was isolated with RNAiso Plus (Takara) according to the manufacturer’s instructions; 600 ng of total RNA was reverse transcribed to cDNA using M-MLV transcriptase (Takara) and oligo (dT) primer (Takara), respectively. cDNA was subsequently subjected to SYBR Green-based real-time PCR using an QuantStudio™ 6 Flex Real-Time PCR Systems (Applied Biosystems, Alameda, CA, USA). Primers used for amplification were as follows: Cpt-1, 5′-TTCACTGTGACCCCAGACGGG-3′ and 5′-AATGGACCAGCCCCATGGAGA-3′; 11β-HSD1, 5′-CCTTGGCTGGGAAAATGACC-3′ and 5′-CTATGAGGCCAAGGACACAGAGAG-3′; FABP3, 5′-CCCCTCAGCTCAGCACCAT-3′ and 5′-CAGAAAAATCCCAACCCAAGAAT-3′; IL-6, 5′-TCCAGCCAGTTGCCTTCTTGG-3′ and 5′-TCTGACAGTGCATCATCGCTG-3′; IL-10, 5′-GCCCTTTGCTATGGTGTCCTTTC-3′ and 5′-TCCCTGGTTTCTCTTCCCAAGAC-3′; β-actin, 5′-AGAGGGAAATCGTGCGTGAC-3′ and 5′-CAATAGTGATGACCTGGCGT-3′. The gene expression was normalized relative to the expression of the control gene (β-actin). The fold change of relative mRNA expression was calculated using the 2^−∆∆Ct method.

Total RNAs as described for RNA-Seq library construction were used for qRT-PCR. Reverse transcription was performed using 1 μg total RNA for each sample and 200 U M-MLV Transcriptase (TaKaRa) in a 10 μl volume. The reaction was conducted at 70 °C for 10 min, 42 °C for 60 min and 70 °C for 15 min. The resulting cDNA was diluted to 800 μl with sterile water. The qRT-PCR was conducted in triplicate reactions using the BIO-RAD CFX system (BIO-RAD). Gene-specific primers were designed by Primer3 (http://bioinfo.ut.ee/primer3/), and the primers for randomly selected 28 genes used in this study are listed in Additional file 16. The primers for validation of all the genes that described in the result part are listed in Additional file 17.
The 40S ribosomal protein S8 (c42687_g2) was used as the internal control for qRT-PCR because the expression level of this gene is relatively stable in samples of different tissues and different time points. The PCR was conducted in a 20 μl volume containing 4 μl diluted cDNA, 250 nM forward primer, 250 nM reverse primer, and 1 × SYBR Premix Ex Taq II (TaKaRa) using the following conditions: 95 °C for 3 min, 40 cycles of 95 °C for 15 s, 59 °C for 15 s and 72 °C for 15 s. Melting curve analyses were performed to verify the specificity using the Bio-Rad CFX Manage software. The relative expression levels were calculated using the 2^-ΔΔCT method [65 ].

Total RNA was extracted with an RNeasy Extraction kit (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using M-MLV transcriptase (TaKaRa Biotechnology, Dalian, China) after DNase I treatment according to the manufacturer’s instructions. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was then conducted to measure the expression levels of the 23 BnCKXs in various tissues of ZS11. After analyzing the RNA-Seq data and the expression patterns of all BnCKXs in ZS11, qRT-PCR was also conducted to measure the expression of selected genes (BnCKX5-1, BnCKX 5-2, BnCKX 6-1 and BnCKX7-1) in seeds and silique pericarps at different stages in B. napus plants with extremely long versus short siliques. The qRT-PCR was performed as described by Ma [58 (link)]. Gene-specific primer pairs for the 23 BnCKXs were designed using Primer Premier 6.0 (Table S2) [59 (link)]. Melting curve analysis and agarose gel electrophoresis were performed to confirm the specificity of the product. Three biological replicates with three technical replicates were performed for each reaction. The BnActin7 gene served as an endogenous reference gene. The calculation of the gene relative expression levels followed the 2^−ΔΔCT method described by Livik and Schmittgen. [60 (link)]

Total RNA used for quantitative RT-PCR (qRT-PCR) analysis was extracted from the different developmental stages of the seeds of the two pea cultivars with three biological replicates using the same method as described above. For qRT-PCR, approximately 3 μg of total RNA was digested using RNase-free DNase I (TaKaRa, Kyoto, Japan) at 37°C to remove any genomic DNA. The digested RNA was then reverse-transcribed to cDNA using MMLV transcriptase (TaKaRa, Kyoto, Japan) in a 20 μl reaction. The cDNAs were then used as templates in qRT-PCR reactions using the SYBR Green Master Mix (TaKaRa, Kyoto, Japan) with gene-specific primers. The reactions were performed on an ABI StepOnePlus™ cycler (Applied Biosystems, USA). The two-step quantitative RT-PCR program began at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. Fluorescent signals were collected at each polymerization step. Expression was calculated as 2^−ΔΔCt (Livak and Schmittgen, 2001 (link)) and was normalized to that of the control gene β-tubulin (Die et al., 2010 (link)). The primers used for qRT-PCR are listed in Supplementary Table S1. The correlation between log2 normalized qRT-PCR and RPKM values was plotted using Origin 8 (OriginLab, USA).

NCI-H460 cells were seeded in 24-well plates at 1 × 10⁵ cells per well overnight at 37 °C. The medium was then removed, and the cells were treated with 300 nM syn-RA16 or scrambled RNA (SCAP) in fresh medium for series incubation time (0, 15, 30, 60, 120, 240, 480, and 960 min). After incubation, cells were rinsed three times with DPBS, and then collected for RNA extraction using TRIzol reagent (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s protocol. The extracted total RNAs were first treated with DNase I to eliminate DNA contamination and quantified using One Drop Spectrophotometry (Hong Kong, China). Next, 500 ng of DNase I-treated RNA was reverse-transcribed into DNA using M-MLV transcriptase (TaKaRa, Dalian, China). Real time PCR was performed with aptamer primers and Power SYBR Green Master Mix (Life Technologies) according to the manufacturer’s protocol, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH, primers sets from Sangon Technologies, Shanghai, China) was amplified for normalization. Quantitative PCR data were analyzed using the StepOnePlus™ Real-Time PCR system (Applied Biosystems). The relative RNA levels with different incubation time were calculated by the 2^−ΔΔCT method using GAPDH as a control.