Smarter pcr cdna synthesis kit

The anthers were removed from the buds and total RNA was extracted from these samples using an RNAprep Pure Kit (DP441, Tiangen, Beijing, China). More than 20 μg of RNA was extracted from each group of samples and was used to construct cDNA libraries. The quality and quantity of the RNA samples were assessed using Qubit and Agilent 2100. A Clontech SMARTer PCR cDNA Synthesis Kit was used to construct the Iso-Seq library. The Iso-Seq library construction started with the mRNA purified with Oligo(dT) magnetic beads (NEB) and cDNA was synthesized by using the SMARTer PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, USA) according to the Iso-Seq protocol (Pacific Biosciences, Menlo Park, CA, USA). Then, cDNA was amplified using KAPA HiFi DNA Polymerase (Roche, Basel, Switzerland) for 12 cycles followed by purification and size selection. The BluePippin Size Selection System protocol was performed by following the instructions described by Pacific Biosciences (PN 100-092-800-03). The insert size of the library was detected using Agilent 2100 to ensure the quality of libraries. Short-read RNA-Seq libraries were prepared using TruSeq stranded RNA Library Preparation kits and the supplied protocol (Illumina, San Diego, CA, USA) and sequenced on a HiSeq2500 platform using v2 sequencing chemistry to generate 2 × 150 paired-end reads.

Copy DNAs were generated from polyA(+) RNA fractions using SMARTer PCR cDNA Synthesis Kits (Clontech) according to ‘PacBio Isoform Sequencing (Iso-Seq) using Clontech SMARTer PCR cDNA Synthesis Kit and No Size Selection’ protocols. Samples from different infection time points (1-, 4-, 8-, and 12-h p.i.) were mixed for RSII library preparation, whereas 1-, 2-, 3-, 4-, 6-, and 8-h p.i. samples were mixed for Sequel sequencing. Samples of cDNA were prepared from rRNA-depleted RNA mixtures from 1-, 4-, 8-, and 12-h time points using modified random hexamer primers (Supplementary Table S13) instead of using the oligo(d)T-containing primer provided in the SMARTer Kit. Samples were used for SMRTbell template preparation using the PacBio DNA Template Prep Kit 1.0.

Complementary DNA was generated from polyA(+) RNA fractions using SMARTer PCR cDNA Synthesis Kits (Clontech, Mountain View, CA, USA) according to PacBio Isoform Sequencing (Iso-Seq) using Clontech SMARTer PCR cDNA Synthesis Kit and No Size Selection protocols. The samples from 1, 2, 3, 4, 6, and 8 h p.i. were used individually to produce libraries for Sequel sequencing. The samples were used for SMRTbell template preparation using the PacBio DNA Template Prep Kit 1.0 (Menlo Park, CA, USA).

For PacBio SMRT sequencing, a pooled RNA sample was mixed from 12 RNA samples used for Illumina RNA-seq with equal amounts. The Isoform Sequencing (Iso-Seq) library was generated following the Iso-Seq protocol using the Clontech SMARTer PCR cDNA Synthesis Kit (Takara, Dalian, China) and the BluePippin Size Selection System protocol which is described by Pacific Biosciences (PN 100–092–800-03).

Equimolar rations of 38 samples were pooled together. Total RNA (1 μg) was reverse-transcribed into cDNA using the SMARTer™ PCR cDNA synthesis kit (Takara Biotechnology, Dalian, China) and optimized to prepare high-quality and FL cDNAs. Subsequently, size fractionation (0.6–1, 1–2 and >2 kb) was conducted using the BluePippin™ Size-Selection System (Sage Science, Beverly, MA). Another amplification was performed using 12–14 PCR cycles. Large-scale PCR products were purified with AMPure PB magnetic beads. Each SMRTbell library was constructed using selected cDNA (500 ng) with the Pacific Biosciences DNA Template Prep Kit 2.0. The SMRTbell templates were bound to polymerases using the DNA/Polymerase Binding Kit P6 and v2 primers. The polymerase-bound template was bound to zero-mode waveguide using Magbeadbingding kit (part 100-133-600). A total of 20 SMRT cells, composed of three SMRTbell libraries (0.6–1 kb: 7 cells; 1–2 kb: 7 cells; >2kb: 6 cells), were prepared on the Pacific Bioscience RS II platform by Frasergen Inc. (Wuhan, China) using C4 reagents with 240 min movies.

Total RNA was isolated from snap‐frozen hearts using RNAiso Plus (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) according to the manufacturer's protocol. First‐strand cDNA was synthesized from RNA using a SMARTer PCR cDNA Synthesis Kit (TaKaRa Bio Inc.). Real‐time quantitative RT‐PCR was performed with a Step One Plus Real‐Time PCR system thermocycler (Thermo Fisher Scientific, Waltham, MA, USA) using SYBR Premix Ex Taq (Tli RNaseH Plus; TaKaRa Bio Inc.). All the primer sequences are listed in Table 1. The relative expression level of specific mRNA was determined by the comparative cycle threshold (C_T) method (2−ΔΔCT) normalized to endogenous control GAPDH gene.