Bluepippin size selection system

Full-length cDNAs were synthesized using a SMARTer™ PCR cDNA Synthesis Kit (Takara Clontech Biotech, Dalian, China) with mRNA templates. The cDNAs were fractionated and selected using a BluePippin^TM Size Selection System (Sage Science, Beverly, MA, USA), resulting in cDNA libraries of different-sized fragments (1–2 k, 2–3 k, and 3–6 k). cDNA libraries were then generated using a Pacific Biosciences DNA Template Prep Kit 2.0 (Menlo Park, CA, USA) and sequenced on a PacBio RS II platform (Menlo Park, CA, USA). Nonchimeric full-length sequences were identified from reads of inserts (ROIs) on the basis of the presence of poly(A) tail signals, 5′ adapter sequences, and 3′ adapter sequences. Then full-length transcripts were clustered into consensus transcripts according to their sequence similarity, using iterative isoform-clustering. Non-full-length transcripts were also clustered and all consensus transcripts were polished using Quiver software (http://www.pacbiodevnet.com/quiver/, accessed on 2 April 2021) [39 (link)]. Low-quality transcript sequences identified by Quiver were corrected by Illumina transcriptome data. Finally, high-quality sequences and low-quality sequences were removed redundancy using CD-HIT [40 (link)].

Approximately 40 g of fresh leaf tissue was collected from an advanced inbred line (>F₁₁) of the winter type oilseed rape accession “Express 617.” DNA libraries of 350 bp, 450 bp, 2 kbp, 5 kbp, and 10 kbp were constructed and subjected to paired-end sequencing on the Illumina HiSeq 2000 platform. The 20-kb SMRTbell library was prepared using SMRTbell Template Prep Kit 1.0-SPv3, where the qualified high-molecular weight DNA were fragmented to approximately 20 kb, followed by damage repair, end repair and adapter ligation. Size selection was then performed using BluePippin^TM Size-Selection System (Sage Science, Beverly, MA, United States). The quality of purified library was checked using Qubit (Invitrogen) and Advanced Analytical Fragment Analyzer (AATI). The SMRTbell-Polymerase Complex was prepared using Sequel^TM Binding Kit 2.0 and sequenced on Sequel SMRT Cell 1M v2. A 6 h movie using the Sequel Sequencing kit 2.0. 10x Genomics libraries also constructed and sequenced on the Illumina platform to produce GemCode linked reads. All sequencing described above was outsourced to Novogene, Co., Ltd. (China). Raw reads obtained were deposited to the NCBI Short Read Archive (PRJNA587046). Sequencing depths obtained for each library are recorded in Supplementary Table S1.

One microgram for each RNA sample was equally pooled together and prepared for PacBio SMRT library construction. Full-length cDNA was synthesized by use of the Clontech SMARTer PCR cDNA Synthesis Kit (TaKaRa, Dalian, China), and cDNA fraction and length selection (<4 kb and >4 kb) was performed using the BluePippin^TM Size Selection System (Sage Science, Beverly, MA, United States). Then, one SMRT bell library was generated using the Pacific Biosciences DNA Template Prep Kit 2.0 (Pacific Biosciences, CA, United States) according to the standard method. All the samples were pooled onto one SMRT cell library, so SMRT data of different tissues were not available, and tissue-specific analysis of isoforms was not possible. Finally, SMRT sequencing was performed on the Pacific Bioscience Sequel platform.

Total RNA was extracted using the Plant RNA Kit (OMEGA, Georgia, USA, No. R6827–01). Equal amounts of RNA collected from ‘Four season’ plants at each sampling day (0, 14th day) were pooled together. The quantity and integrity of RNA samples were assessed in the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Delaware, USA) and in the 2100 Bioanalyzer (Agilent Technologies, California, USA), respectively. Qualified RNA samples were then used for constructing cDNA libraries. The SMARTer PCR cDNA Synthesis Kit (TaKaRa, Dalian, China) was utilized to synthesize full-length cDNA and cDNA fraction and length selection (1–2 kb, 2–3 kb, and > 3 kb) were performed using the BluePippin^Tm Size Selection System (Sage Science, Massachusetts, USA). These three SMRT libraries were generated at Biomarker Technology Co. (Biomarker, Beijing, China) using the Pacific Biosciences DNA Template Prep Kit 2.0. (Pacific Biosciences, California, USA) according to the standard protocol. SMRT sequencing was then performed on the Pacific Bioscience RS II (Pacific Biosciences, California, USA) platform at Biomarker Technology Co. (Biomarker, Beijing, China) using the protocol provided by the manufacturer.

First, the qualified RNA samples were equally pooled together. Then, full-length cDNA synthesis was conducted using the SMARTer PCR cDNA Synthesis Kit (Clontech, USA). Next, the BluePippinTM Size Selection System (Sage Science, USA) was applied for cDNA size fractionation and length selection. Subsequently, the PB library was prepared using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, USA). Lastly, the PB Sequel platform was used for SMRT sequencing.