Vahts universal v6 rna seq library prep kit

Total RNA was extracted from the muscle tissues with the Trizol reagent (Takara Bio, Otsu, Japan). RNA purity and quantity were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, from Waltham, MA, USA), and the total RNA was measured using ≥1 μg, OD260/280 in 1.8–2.2, Agilent2100 RIN ≥ 7. Its integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA libraries were then constructed using VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina according to the manufacturer’s instructions. The libraries were finally sequenced using the Illumina NovaSeq 6000 platform, and 150 bp-long paired-end reads were generated. Transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).

Total RNA was extracted from the midgut of each treatment group of BSFL using R6834 Total RNA Kit I. Three replicates were used for each treatment group, with each replicate comprising 15 midgut samples of BSFL. After quality control, VAHTS Universal V6 RNA-seq Library Prep Kit (Illumina®, NR604-01/02) was used to select different index tags for library construction. The qualified libraries were sequenced using the HiSeq sequencing platform.

Total RNA from liver tissue samples was extracted with TRIzol (Catalog No. 15596026; ThermoFisher, USA) according to the standard isolation protocol. The RNA libraries for sequencing were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Catalog No. NR604-01) with 2 ug total RNA according to the manufacturer’s protocol. In brief, poly(A) + mRNA was enriched from the total RNA using the mRNA Capture Beads, and then the purified mRNA was randomly fragmented into sequences approximately 400 bp in length. A cDNA library was obtained with random hexamer primers. After the end repair of the cDNA fragments, adaptors were added to the other end of the cDNA products, and then the cDNA library was amplified by PCR. After validation by qPCR, libraries were finally sequenced on the Illumina HiSeqXTen platform using the PE150 module. DEGs were identified using the DESeq2 program with the cutoff threshold of P < 0.05 and the absolute value of log2 fold change (log2 FC) > 1. GO (http://www.geneontology.org/) and KEGG enrichment analyses (http://www.genome.jp/kegg) were performed using DEGs as the foreground genes and all genes as the background.

RNA was extracted from tissues by using TIANGEN® RNAprep Pure FFPE Kit (#DP439) according to the reagent protocols. For library preparation of RNA sequencing, a total amount of 500 ng RNA per sample was used as the input material for the RNA sample preparations. Sequencing libraries were generated using Ribo-off® rRNA Depletion Kit (H/M/R) (Vazyme #N406) and VAHTS® Universal V6 RNA-seq Library Prep Kit for Illumina (#N401-NR604) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. The libraries were sequenced on an Illumina platform and 150 bp paired-end reads were generated.

A total amount of 1–3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using VAHTS Universal V6 RNA-Seq Library Prep Kit for Illumina ^®. In order to guarantee the data quality used to analysis, the useful Perl script was used to filter the original data (Raw Data). The reference genomes and the annotation file were downloaded from B. rapa genome v1.5 sequence, B. nigra genome v1.1 sequence and B. oleracea genome v1.0 sequence (http://Brassicadb.cn, accessed on 1 May 2021). Bowtie2 v2.2.3 was used for building the genome index, and Clean Data was then aligned to the reference genomes using HISAT2 v2.1.0 [71 (link)]. The RNA-Seq data was uploaded to the NCBI Gene Expression Omnibus (GEO), and its accession number, GSE201456, may be used to retrieve it.

After conducting comparative analyses of various growth and physiological indicators, we selected four treatment groups for transcriptome sequencing: Control (CK), 250 mg·kg⁻¹ Ni (Ni), 100 μmol·L⁻¹ MT (MT), and 250 mg·kg⁻¹ Ni + 100 μmol·L⁻¹ MT (Ni_MT).
The total RNA was extracted from the frozen leaves of R. typhina seedlings using TRIzol (Invitrogen, Waltham, MA, USA). RNA purity and quantification were evaluated by a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and the RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Next, the transcriptome library was constructed using the VAHTS Universal V6 RNA-Seq Library Prep Kit (Illumina, San Diego, CA, USA) according to the instructions. After the quality of the library was qualified by the Agilent 2100 biological analyzer (Agilent Technologies, Santa Clara, CA, USA), the Illumina Novaseq 6000 (Illumina, San Diego, CA, USA) sequencing platform was used for sequencing to generate a 150 bp double-ended sequence. Transcriptome sequencing and analysis were performed by Shanghai OE Biotechnology Co., Ltd. (Shanghai, China).