Aglient 2100 bioanalyzer

Total RNA was isolated using the Illustra RNAspin Mini kit (GE Healthcare Life Sciences, Buckinghamshire, UK). Approximately 5 × 10⁶ cultured cells were processed following the manufacturer's instructions. Samples were eluted in Ultrapure DNase/RNase-free distilled water, which was provided in the kit. RNA samples were quantified using ultraviolet spectroscopy (NanoDrop, Wilmington, DE) and were further assessed for RNA integrity (RIN) on the Aglient 2100 Bioanalyzer (Santa Clara, CA) using the RNA Nano-chip Kit. RNA samples with RIN values of seven or better were used for further analysis.

The miRNeasy® Mini Kit (Qiagen, Hilden, Germany) was used to extract the total RNA from 25–35 individuals per replicate of the collembolans that were fed in groups for each generation. The RNA concentration and integrity was evaluated with Aglient 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).
The cDNA library construction, Illumina sequencing, and de novo assembly of RNA samples were carried out by Hangzhou 1gene Technology Co., Ltd (Hangzhou, China). All cDNA libraries were constructed using the NEBNext®Ultra™ RNA Library Prep Kit for Illumina® (NEB, Ipswich, MA, USA) following the protocol described by the manufacturer. The libraries were sequenced with 150-bp paired-end reads on an Illumina Hiseq 4000 platform (San Diego, CA, USA). After sequencing, the raw reads (about 6 GB of data for each replicate) were filtered to remove adaptor sequences, duplication sequences, and low-quality sequences. The clean reads were de novo assembled into unigenes using Trinity and SOAPdenovo-Trans. Six public databases were used to annotate unigenes with BLASTx or BLASTn (E-value < 10⁻⁵), including NCBI Nr and Nt databases (http://www.ncbi.nlm.nih.gov/), SwissProt (http://www.expasy.ch/sprot/), KEGG (http://www.genome.jp/kegg/), COG (http://www.ncbi.nlm.nih.gov/COG/), and GO (http://www.geneontology.org/).

Total RNA was isolated using the Illustra RNAspin mini kit (GE Healthcare Life Sciences, Buckinghamshire, UK). Approximately 5 × 106 cultured cells were processed following the manufacturer's instructions. Samples were eluted in Ultrapure DNase/RNase-free distilled water provided in the kit. RNA samples were quantified using ultraviolet spectroscopy (NanoDrop, Wilmington, DE) and were further assessed for RNA integrity (RIN) on the Aglient 2100 Bioanalyzer (Santa Clara, CA) using the RNA Nano-chip Kit. RNA samples with RIN values of seven or higher were used for further analysis.

Mouse subcutaneous adipose tissue, hypothalamus, and ovary were homogenized in 1 ml TRI reagent (T9424, Sigma-Aldrich). Total RNA was extracted as per the manufacturer’s instructions and quality was confirmed by Agilent 2100 bioanalyzer with RIN 6–7. Then 1 ng of total RNA was applied to bulk RNA-sequencing library. Sequencing libraries were generated according to Smart-seq3 protocol^{61 (link)}. Briefly, polyA(+) RNA was reverse transcribed by Maxima H-minus reverse transcriptase (Thermo Fisher). The second-strand synthesis was conducted by a template-switching reaction and 12 cycle of PCR was performed for cDNA amplification by KAPA HIFI HotStart polymerase (Roche). Then cDNA was purified by 22% PEG (Sigma-Aldrich) beads. Aglient 2100 BioAnalyzer (Agilent Technologies) was performed to check the quality and quantity of cDNA libraries. Sequencing libraries were generated by tagmenting 200 pg cDNA using Nextera XT Tn5 transposase (Illumina) and amplified for 10 cycles.

Total RNAs from seven Beauveria were extracted using Trizol reagent (Life Technologies, Carlsbad, CA, USA) following the reagent handbook. The obtained RNA samples were subjected to RNase-free DNase I treatment to remove residual DNA. The purity, concentration, and integrity of the RNA were tested using a NanoDrop 2000 spectrophotometer (Implen, CA, USA), Aglient 2100 bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA), and Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA), respectively. The mRNAs were purified from the total RNAs using poly-T oligo-attached magnetic beads and fragmented. Subsequently, cDNA libraries were generated for the quality-checked samples using a Tru Seq RNA Sample Prep Kit following the kit handbook. The successfully generated cDNA libraries were sequenced using using an Illumina NovaSeq 6000 platform (Illumina, Inc., CA, USA).

The RNA Prep Pure Plant kit (Tiangen Biotech, Beijing, China) was used to extract total RNA from the leaf, stem, and root of A. paniculata (three biological replicates per tissue). The concentration and integrity of RNA samples were assessed using an Aglient 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Nine RNA samples from A. paniculata that met the transcriptome sequencing requirements were sent to Nuohe Zhiyuan Biotechnology Co., Ltd, Shanghai, China. for sequencing on the Illumina Novaseq 6000 platform. The raw data were cleaned to remove adapters, N bases, and low-quality reads. The GC percentage, Q20, and Q30 were also calculated. Clean reads were de novo assembled into transcripts and unigenes using Trinity 2.4.0 software [62 (link)]. The blast2go tool [63 (link)] was used to perform functional annotation against various databases, including NCBI non-redundant protein sequences (NR), NCBI nucleoside sequences (NT), KEGG, GO, euKaryotic Ortholog Groups/Clusters of Orthologous Groups of proteins (KOGs), Pfam, and Swiss prot. The iTAK 1.2 software [64 (link)] identified transcription factors (TFs) in A. paniculata.

We placed the adipose tissues in a mortar pre-cooled with liquid nitrogen and quickly ground it to powder. In order to obtain total RNA, we sequentially added 1 mlTRlzol((Invitrogen Life Technologies, Carlsbad, USA), 0.2 ml chloroform, 0.2 ml isopropanol, 75% ethanol and RNase-free water. The integrity of sample RNA was preliminarily determined by 1% agarose gel electrophoresis. To ensure RNA concentration, we used Nanodrop to detect od260 / 280 and od260 / 230 values. We accurately evaluated the integrity, purity and degradation degree of RNA through Aglient 2100 Bioanalyzer. Six cDNA libraries (L_PX_1, L_PX_2, L_PX_3 and L_JN_1, L_JN_2, L_JN_3) were constructed through TruSeq Stranded Total RNA LT Kit with Ribo-Zero TM Gold (RS-122-2301). The libraries building process includes mRNA enrichment, fragmentation, reverse transcription, double-stranded cDNA synthesis, linker, DNA purification and PCR amplification. To test the length and quality of the library, we added samples to the Agilent 2100 biological analyzer. After the quality inspection is qualified, samples were sequenced by Illumina HiSeqTM2500 platform.