Agilent 2100

As shown in the following Figure 1, the workflow of mRNA sequencing includes sample preparation, library construction, library quality control, and sequencing.
Purity, concentration and integrity of RNA sample were examined by NanoDrop, Qubit 2.0, Agilent 2100, etc. Only RNA with good quality could move on to following procedures. (1) mRNA was isolated by Oligo(dT)-attached magnetic beads. (2) mRNA was then randomly fragmented in fragmentation buffer. (3) First-strand cDNA was synthesized with fragmented mRNA as template and random hexamers as primers, followed by second-strand synthesis with addition of PCR buffer, dNTPs, RNase H, and DNA polymerase I. Purification of cDNA was processed with AMPure XP beads. (4) Double-strand cDNA was subjected to end repair. Adenosine was added to the end and ligated to adapters. AMPure XP beads were applied here to select fragments within size range of 300–400 bp. (5) cDNA library was obtained by certain rounds of PCR on cDNA fragments generated from step 4. In order to ensure the quality of library, Qubit 2.0 and Agilent 2100 were used to examine the concentration of cDNA and insert size. Q-PCR was processed to obtain a more accurate library concentration. Library with concentration larger than 2 nM is acceptable. The qualified library was pooled based on pre-designed target data volume and then sequenced on Illumina sequencing platform.

A total of 500 pairs of male and female D. tarsalis were collected and combined respectively. Total RNA was extracted using an assay kit and Nanodrop was used to measure RNA purity and concentration. Agilent 2100 was used to measure RNA integrity and qualified samples had an OD (260/280) of 1.8–2.2, RIN ≥ 7.5, and nucleic acid electrophoresis was used to detect the integrity of RNA. Qualified samples were used for library construction. The SMARTer™ PCR cDNA Synthesis Kit (Clontech, Palo Alto, CA, USA) was used to synthesize full-length cDNA of mRNAs. PCR was used to amplify full-length cDNAs, and end-terminal repair was carried out on full-length cDNAs. Then, SMRT dumbbell adapters (SMRTbell Template Prep Kit) were ligated to cDNAs and exonuclease digestion was carried out to obtain sequencing libraries. After library construction was completed, Qubit2.0 was used for accurate quantitation and Agilent 2100 was used to measure the size of the libraries. After the library quality control was confirmed to meet expectations, PacBio RS II was used for full-length transcriptome sequencing, which was carried out by Biomarker Technologies Co. Ltd. (Beijing).

The total RNA was extracted from the three biological replicates of WP, GP, WL, and GL samples of water dropwort, respectively. The purity and quality of RNA were estimated by Nanodrop, Agilent 2,100, and electrophoresis. A total of 12 Illumina RNA sequencing libraries was constructed using the NEBNext^® Ultra™ RNA Library Prep Kit (NEB, Massachusetts, United States) according to the manufacturer’s protocols. The mixed RNA samples, F01 (WP and WL) and F02 (GP and GL), were used to construct the PacBio Iso-Seq library. The full-length cDNA was synthesized by SMARTer™ PCR cDNA Synthesis Kit (TaKaRa, Dalian, China) following the operating instructions. Bluepippin (Sage Science Beverly, MA, USA) was used to screen full-length cDNA fragments and construct cDNA libraries with different sizes (1–2, 2–3, and >3 kb). The constructed libraries were evaluated by Qubit 2.0 Fluorometer and Agilent 2,100. The SMRT sequencing and NGS was performed with Pacific Bioscience RS II (Pacific Biosciences, California, United States) platform and Illumina HiSeq4000 platform (San Diego, CA, United States) at Biomarker Technology Co. (Biomarker, Beijing, China), respectively.

Total RNA was extracted using RNAiso Plus (TaKaRa Bio, Beijing, China) according to the manufacturer’s protocol. The mRNA was enriched with Oligo(dT) magnetic beads and fragmented with fragmentation buffer after being assessed for purity and integrity with NanoDrop (Thermo Fisher Scientific, Wuhan, China) and Agilent 2100 (Agilent, Beijing, China).
The first strand cDNA was synthesized using random hexamers, and during the second strand cDNA synthesis, dTTP was replaced by dUTP to indicate strand specificity. The library was built after purification (AMPure XP beads), adaptor (including barcode) ligation, UDG degradation, and PCR amplification. The Agilent 2100 was used to inspect the quality of these libraries before they were used for paired-end sequencing on the HiSeqTM4000 (Illumina, Beijing, China). Under the accession number CRA004201, the clean data were uploaded to the GSA database: http://bigd.big.ac.cn/gsa (accessed on 18 February 2023) [37 (link)].