Rnapure total rna kit

Thirty workers (Pirk et al., 2015) from each colony were crushed to a fine powder in liquid nitrogen and used for RNA isolation using the RNApure Total RNA Kit (Aidlab Biotechnologies Co. Ltd.), according to the manufacturer's protocol. cDNA synthesis was conducted using 800 ng RNA and ReverTra Ace qPCR RT Master Mix (Toyobo), according to the manufacturer's instructions.

Fifteen and forty pairs of antennae of E. scrobiculatus and E. brandti were excised separately, and total RNA of female antennae (FA) and male antennae (MA) were extracted using the RNApure Total RNA Kit (Aidlab, Beijing, China). For each species and sex, data were obtained for three independent biological replicates, for a total of 12 samples. RNA was quantified using a NanoDrop 8000 (Thermo, Waltham, MA, United States). cDNA library construction and Illumina sequencing were performed at Bionova Biotechnology Co., Ltd. (Beijing, China). RNA quality was assessed by 1% agarose gel electrophoresis and analyzed using the 2100 Bioanalyzer (Agilent, Santa Clara, CA, United States). RNA was digested by DNase I to remove the DNA, and mRNA was enriched using oligo d(T). The mRNA was fragmented at a high temperature and reverse-transcribed. The resultant cDNA was subjected to purification, end repair, A-tailing, adapter ligation, and PCR amplification. The quality and quantity of the library were then evaluated using the Bioanalyzer 2100 and ABI StepOnePlus Real-Time PCR system (Applied Biosystems, Forester City, CA, United States), respectively. The qualified library was then used for high-throughput sequencing. These libraries were pair-end sequenced using the PE150 strategy on the Illumina HiSeq X Ten platform.

Total RNA extraction and RT-qPCR analysis were performed according to Li et al. with minor modifications (54 (link)). Mycelia were harvested after 5 days of cultivation. RNA was isolated according to the manufacturer’s instructions by using an RNApure Total RNA Kit (Aidlab Biotechnologies Co., Ltd, Beijing, China). Briefly, after treatment with RNase-free DNase I, RNA samples were qualified and quantified by using a NanoDrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA) and Agilent 2100 Bioanalyzer (Agilent). First-strand cDNA was synthesized according to the manufacturer’s instructions (Transgen, Beijing, China). SYBR green qPCR Master Mix (Transgen) and the QuantStudio 6 Flex (Applied Biosystems, Carlsbad, CA, USA) qPCR system were used to determine gene expression. The qPCR primers are listed in Table S5.

The expression levels of selected DEmRNAs, DEmiRNAs, DEcircRNAs, and DElncRNAs were validated by qRT-PCR. Total RNAs and small RNAs of ‘Ant’ and ‘Mix’ were extracted using an RNApure Total RNA Kit (Aidlab, RN03, China). RNA was reverse transcribed using an Evo M-MLV RT Kit II (Accurate Biotechnology, AG11711, China). Oligo dT primers and random 6-mer primers were used to construct the 1st strand cDNA for quantitative analysis of mRNAs and lncRNAs. The miRNAs were reverse-transcribed using the downstream primers of U6 endogenous reference gene and the specific stem-loop primers. The circRNAs were reverse-transcribed using the qRT‒PCR downstream primers and random 6-mer primers. qRT-PCR was performed using a SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology) with an ABI 7300 instrument (Thermo Fisher Scientific, Waltham, MA, USA). The qRT-PCR procedure was as follows: 50 °C for 2 min; 95 °C for 30 s; 40 cycles at 95 °C for 5 s and 60 °C for 30 s; 95 °C for 15 s; 60 °C for 1 min; and 95 °C for 15 s. U6 was used as an endogenous control for the miRNAs. Actin was used as an endogenous control for circRNAs, lncRNAs, and mRNAs. All qRT-PCR reactions were performed in three technical replicates and three biological replicates. The primer sequences are listed in Table S12. RT is the specific stem-loop primers used to amplify miRNAs.

Petals were selected from the 10 phenotypically similar plants of ‘FT’, pdm, and pdm + JA, respectively. The extraction of total RNA and cDNA synthesis were respectively conducted using an RNApure Total RNA Kit (Aidlab, Beijing, China) and a HiScript II Q Select RT SuperMix for qPCR (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Equal amounts of total RNA from three biological replicates of ‘FT’, pdm, and pdm + JA were respectively pooled for RNA-Seq library construction, which we designated as FT-1, FT-2, and FT-3; pdm-1, pdm-2, and pdm-3; and pJA-1, pJA-2, and pJA-3.
The quantity and purity of total RNA were determined using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, Carpinteria, CA, US) with RIN number > 7.0. The nine cDNA libraries were sequenced with 6 G depth using the Hiseq4000-PE150 sequencing platform (Illumina, San Diego, CA, USA) by Novogene (Beijing, China).

The transcriptome of different stages of TRW (accession number: PRJNA689057) were already sequenced by Wu et al. (2016 (link)), so we prepared samples for the RNA sequencing of TTW in this study. TTW adults, larvae, pupae, and TRW adults were collected from the Pingluo County, Ningxia Autonomous Region, China. About 100 of the TTW adults were being reared at the Forest Protection Laboratory of Beijing Forestry University for oviposition. Each pair of adults (a male and female) was fed with A. altissima sticks in a plastic box with a diameter of 3.5 cm at 25 ± 1°C and 75 ± 1% relative humidity. Two-day-old eggs of TTW were removed with a fine brush and placed on a Petri dish lined with soaked filter paper, in preparation for RNA extraction. The fifth-instar larvae were selected for RNA extraction because of their strong foraging ability. Total RNA of a single adult, single 4-day pupa, single fifth-instar larva, and 40 eggs was extracted with the RNApure Total RNA Kit (Aidlab, Beijing, China). The total RNA of 40 pairs of antennae, 40 proboscises, 10 heads (without antennae and proboscises), two groups of legs (one included a pair of forelegs, midlegs, and hindlegs), and one abdomen (without thorax) was extracted with the methods above. Three biological repetitions were used for all RNA extractions.