Clontech smarter pcr cdna synthesis kit

Total RNA from different tissues was mixed at equal ratios. Poly(A) RNA was isolated from total RNA using Dynal oligo(dT)25 beads (Life Technologies, USA) and used for construction of the Iso-Seq library. The first cDNA strand was synthesized from purified polyA RNAs using the Clontech SMARTer PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, USA). After PCR optimization, large-scale PCR was performed to synthesize the second cDNA strand without size selection. Equimolar mixed libraries of unfiltered fragments and > 4 kb fragments were prepared with the SMRTbell Template Prep Kit 1.0. Sequencing was performed on a PacBio Sequel platform. A total of four SMRT cells were utilized in this study.

SV Total RNA Isolation System (Promega Corporation, Madison, WI, USA) was used to isolate the B. odoriphaga eggs, second instar larvae, fourth instar larvae, pupae, and adult total RNA according to the manufacturer’s protocols, after which possible residual genomic DNA was removed with deoxyribonuclease (DNase I: Fermentas Inc., Burlington, ON, Canada). The RNA integrity was determined using Agilent RNA 6000 Nano Reagents Port 1 (Santa Clara, CA, USA) on a Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) (Table S1). Total RNA from different developmental stages was mixed as one sample for cDNA library construction. cDNA synthesis and library construction was conducted using a Clontech SMARTer PCR cDNA Synthesis Kit (catalogue number: 634925) (Clontech, Mountain View, CA, USA). Sequencing was conducted on the Pacific Biosciences RS II platform (Pacific Biosciences, Menlo Park, CA, USA). BluePippin (Sage Science, Beverly, MA, USA) was used for selection of cDNA sequences ranging in size from 1 to 2 kb, 2 to 3 kb, and 3 to 6 kb. Three, three, and two SMRT (Single-molecule real-time sequencing) cells were used to sequence the 1–2 kb, 2–3 kb, and 3–6 kb libraries, respectively, and the reads were deposited in the NCBI (National Center for Biotechnology Information) Sequence Read Archive database under the accession numbers SRR8903502, SRR8903501, and SRR8903503.

Total RNA was extracted from the leaf samples. A Nanodrop 2000 (Thermo Fisher Scientific, Austin, TX, USA) was used to determine the concentration and purity of the extracted RNA. Subsequently, 1% agarose gel electrophoresis was conducted to evaluate the genomic contamination, purity, and RNA integrity of the samples. An Agilent 2100 (Agilent Technologies, Palo Alto, CA, USA) was used to determine the RNA integrity number (RIN) value. The full-length cDNA of the extracted mRNA was synthesized using a Clontech SMARTer PCR cDNA Synthesis Kit (Clontech Laboratories, Frederick, MD, USA). Primers with Oligo dT were used to pair with the A-T bases at the polyA region for the reverse transcription of the mRNA to obtain cDNA. The full-length cDNA was amplified by PCR, and the product was purified using PB magnetic beads to remove fragments of cDNA that were less than 1 kb. The end of cDNA was repaired, and the fragments were connected with the SMRT dumbbell connector. The unconnected fragments were digested using exonuclease and purified using PB magnetic beads to obtain the sequencing library. A Qubit 3.0 fluorometer (Invitrogen, Carlsbad, CA, USA) was used to accurately quantify the library, and an Agilent 2100 was used to determine the library size. The DNA was sequenced after the library size met the criteria.

Total RNA was extracted from the bulb, leaf, root, and stem samples (4 tissues × 3 biological repeats) of F. hupehensis using the Trizol RNA extraction kit (Invitrogen, Carlsbad, CA, USA). The NanoDrop 2000 spectrophotometer was used to check the RNA concentration and purity (Thermo Fisher Scientific, Wilmington, DC, USA). The extracted RNAs were combined to provide a full RNA. Using a magnetic d(T) bead binding procedure, mRNA was isolated from total RNA and transcribed to cDNA with the use of a Clontech SMARTer PCR cDNA Synthesis Kit (Clontech Laboratories, Inc., CA, USA). In selecting PCR products, the BluePippinTM Size Selection Method (Sage Science, Beverly, MA, USA) was used and fragments of 0.5–6 kbs were retained. Long-scale PCR was then used to enhance the cDNA. The cDNA ends were repaired and the sequence adapters linked to cDNA were ligated. Bell libraries for the SMRT templates were developed with cDNA and sequenced on the PacBio Sequel platform with P6-C4 chemistry, 10-h film times. The Gene Denovo Biotechnology Company (Guangzhou, China) performed all sequencing work. Raw reads were further filtered to attain clean reads by exclusion of adaptors, reads with more than 10% of unknown nucleotides, and poor quality reads. Clean reads, Q30, and GC content were computed.

Equal amounts of RNAs from different hairy root groups (SA_0h, SA_8h, and SA_24h) were pooled to provide the total RNA of P. tunicoides. The mRNA was enriched using the Oligo d(T) magnetic beads, then reverse transcribed to cDNA using Clontech SMARTer PCR cDNA Synthesis Kit (Clontech, United States). Amplification of double-stranded cDNA was followed by size selection using the BluePippin system (Sage Science, United States), and fragments of 1–6 kb were retained. The full-length cDNA was generated, and the cDNA ends were repaired and ligated to sequencing adapters. SMRTbell template libraries were obtained and subsequently sequenced on the PacBio Sequel RS sequencing instrument.

Before establishing the library, the quality of the total RNA was determined. Agarose gel electrophoresis was used to analyze the degree of degradation of RNA and possible contamination. A Nanodrop nucleic acid quantifier was used to detect the purity of RNA (OD260/280 ratio), a Qubit RNA assay was used to accurately quantify the RNA concentration, and an Agilent 2200 TapeStation was used to accurately detect the integrity of the RNA. The Clontech SMARTer^® PCR cDNA Synthesis Kit (Clontech Laboratories, 634926) and the BluePippin Size Selection System protocol, as described by Pacific Biosciences (PN 100-092-800-03), were used to prepare the Iso-Seq library according to the Isoform Sequencing protocol (Iso-Seq).

According to the PacBio Isoform Sequencing protocol, the Clontech SMARTer PCR cDNA Synthesis kit (Clontech, Mountain View, CA, USA) was used for cDNA synthesis and library construction. The full-length cDNA Iso-Seq template was sequenced in two SMRT cells on a PacBio Sequel System after purification, size selection, re-amplification and SMRTbell template preparation. Size selection was performed via a BluePippin (Sage Science, Beverly, MA) to construct a cDNA library of size > 4 kb. The same amount of non-selected cDNA and > 4 kb cDNA were combined to form Iso-Seq library for SMRT sequencing.