Mrnaseq sample preparation kit protocol

Total RNA was extracted from cells with TRIzol reagent (Invitrogen) and total RNA was quantified and analyzed by Agilent Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent, CA, USA) with an RNA integrity number >7.0. Poly‐T oligo‐attached magnetic beads were used to purify poly(A) mRNA by two rounds of purification. The mRNA was fragmented and reverse‐transcribed to construct the cDNA library using the mRNASeq sample preparation kit protocol (Illumina, San Diego, USA); and the cDNA library was paired‐end sequenced on an IlluminaHiseq4000 following the protocol. Clean reads were mapped to the hg38 human genome by HISAT2 (version 2.2.0) and sorted by samtools (version 1.9). Mapped reads were visualized with the Integrative Genomics Viewer (IGV). StringTie (1.3.0) was used to reconstruct transcripts, and edgeR was used to analyze differential gene expression. Genes with |Fold change| > 2 and p <0.05 were considered to be differentially expressed. The short‐reads RNA‐seq data were deposited in the Gene Expression Omnibus (GEO) under accession code GSE223091.

For circRNAs and mRNA, the total RNA samples were treated with an Epicenter Ribo‐Zero Gold Kit (Illumina) to remove rRNA before constructing RNA‐seq libraries. The samples were fragmented and then synthesized as first‐ and second‐strand complementary DNA (cDNA) with random hexamer primers, dNTPs and DNA polymerase I using a PrimeScript RT reagent Kit (TaKaRa). The cDNA fragments were treated with T4 DNA polymerase to repair the ends and Klenow DNA polymerase to add ‐A and adapters at the 3′ end of the DNA fragments. The cDNA products were purified with AMPure XP beads and then subjected to PCR amplification. Then, the cleaved RNA fragments were reverse‐transcribed to create the final cDNA library in accordance with the mRNA‐Seq sample preparation kit protocol (Illumina). Then, we performed paired‐end sequencing on a HiSeq 4000 sequencing system (Illumina) following the manufacturer's recommended protocol.
For miRNAs, approximately 1 μg of total RNA was used to prepare a small RNA library according to the TruSeq Small RNA Sample Prep Kit protocol (Illumina). Then, we performed single‐end sequencing (1 × 50 bp) on an Illumina Hiseq 2500 sequencing system following the manufacturer's recommended protocol.

The adult N. cincticeps were frozen, anesthetized on ice, and their salivary gland (Sg), midgut (Mg), Malpighian tube (Mt), and residual body (Rb) were dissected on ice-cold sterile phosphate buffer solution (PBS, pH = 7.2) treated with 0.1% diethylpyrocarbonate under a stereomicroscope. Total RNA from each tissue was prepared with a Trizol Reagent Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. The degradation and contamination of total RNA were analyzed with Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent, Palo Alto, CA, USA). After extracting total RNA, mRNA was purified from total RNA (5 μg) using poly-T oligo-attached magnetic beads. The isolated mRNA was fragmented using a fragmentation buffer. These fragments were then used as templates for reverse transcription, resulting in the final cDNA library. The mRNASeq sample preparation kit protocol (Illumina, San Diego, CA, USA) was followed. Subsequently, paired-end sequencing was conducted on an Illumina Hiseq 4000 at LC Sciences, USA, following the vendor’s protocol.