Dna template prep kit 2

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.

Using the TRIzol kit (Invitrogen, CA, USA) and its instructions, total RNA was extracted from 12 centipedegrass leaf samples. Each sample was electrophoresed on an agarose gel to determine its RNA quality. The RNA purity was determined using a NanoPhotometer^® spectrophotometer (IMPLEN, Munich, Germany). Using SMARTer™, the PCR cDNA Synthesis Kit (Pacific Biosciences, San Diego, CA, USA) synthesized the full-length cDNA of mRNA, amplified the full-length cDNA using PCR, repaired the ends of the full-length cDNA, connected the SMRT dumbbell-shaped connector and finally performed exonuclease digestion to obtain the cDNA library. The Pacific Biosciences DNA Template Prep Kit 2.0 was used in the construction of SMRTbell iso-seq libraries. Sequencing was performed on the RS II platform (Pacific Biosciences, San Diego, CA, USA). To construct the cDNA library for SGS, the manufacturer provided a protocol for using the NEBNext^® UltraTM RNA Library Prep Kit for Illumina^® (NEB, Beverly, MA, USA). SGS produced 125 bp paired-end sequence reads (2 × 125 bp) based on qualified libraries applied to Illumina’s NovaSeq 6000 (Pacific Biosciences, San Diego, CA, USA). The Biomarker Technology Co. (Beijing, China) performed the high-throughput sequencing (TGS and SGS) for this study.

Genomic DNA (3 μg) was sheared using the HydroShear Plus (Digilab) and a library was prepared using a DNA Template Prep Kit 2.0 (Pacific Biosciences), according to the manufacturer’s instructions. Sequencing was performed with XL polymerase and a DNA Sequencing Kit C2 (Pacific Biosciences) and three SMRT cells (120 min movies). De novo assembly was performed with Sprai v0.9.5
[26 ] and HGAP v2.1.0
[14 (link)] with default parameters. The contigs from Sprai were circularized with a script in the Sprai package when the script detected a significant overlap between the beginning and end of contigs.