According to the Iso-Seq protocol, 1 µg total RNA was transcribed to generate full-length cDNA using the SMARTer PCR cDNA Synthesis Kit (Clontech, CA, USA). Then, the cDNA was amplified using the advantage 2 PCR kit (Clontech, CA, USA), and PCR products were purified with AMpure PB beads (Beckman Coulter, CA, UAS). Purification was followed by size selection using the BluePippinTM Size Selection System (Sage Science, MA, USA) of the following bins: 1-2, 2-3 and 3-6 kb. The three libraries were then constructed using SMRTbell Template Prep kit (Pacific Biosciences, CA, USA). Before sequencing, the quality of the libraries was assessed by Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) and Qubit fuorometer 2.0 (Life Technologies, CA, USA). Libraries were prepared for sequencing by annealing a sequencing primer and adding polymerase to the primer annealed template. The polymerase-bound template was bound to MagBeads and a total of 6 SMRT cells were sequencing on PacBio RS II platform using P6-C4 chemistry (2 cell each library).
Smrtbell template prep kit
Manufactured by Pacific Biosciences
Sourced in United States, China
The SMRTbell Template Prep Kit is a laboratory equipment product designed for the preparation of DNA samples for use with Pacific Biosciences' sequencing platforms. The kit provides the necessary reagents and protocols to convert DNA samples into SMRTbell templates, which are the required input format for Pacific Biosciences' sequencing technologies.
Automatically generated - may contain errors
Lab products found in correlation
Smarter pcr cdna synthesis kit, by Takara Bio (8 mentions)
Qiaquick gel extraction kit, by Qiagen (8 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (8 mentions)
Transstart fastpfu dna polymerase, by Transgene (6 mentions)
Bluepippin size selection system, by Sage Science (5 mentions)
Ampure pb bead, by Pacific Biosciences (4 mentions)
Smrt cell, by Pacific Biosciences (4 mentions)
G tube, by Covaris (4 mentions)
Pacbio rs 2, by Pacific Biosciences (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Pacbio rs 2 platform, by Pacific Biosciences (2 mentions)
Clontech smarter pcr cdna synthesis kit, by Takara Bio (2 mentions)
Pacbio sequel platform, by Pacific Biosciences (2 mentions)
Sequel instrument, by Pacific Biosciences (2 mentions)
Smrt analysis software, by Pacific Biosciences (2 mentions)
Pacbio rs 2 sequencing platform, by Pacific Biosciences (2 mentions)
Qubit 4.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Femto pulse system, by Agilent Technologies (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
62 protocols using smrtbell template prep kit
1
Iso-Seq: Full-Length cDNA Sequencing
2
Full-Length Transcriptome Sequencing of cDNA
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First-strand cDNA was synthesized using a SMARTer™ PCR cDNA Synthesis Kit (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instructions. After subsequent PCR amplification, quality control, and purification, the cDNA products and the Pacific Biosciences SMRTbell Template Prep Kit were used to construct a library. Qubit2.0 was then used to quantify the library accurately, and Agilent 2100 was used to ensure that the library size was consistent with expectations. Finally, the library was subjected to full-length transcriptome sequencing on the PacBio RS II platform by Beijing Biomarker Technologies Co., Ltd (Beijing, China).
3
PacBio Whole Genome Sequencing and Assembly
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A genomic DNA library was constructed using a SMRTbell Template Prep kit (Pacific Biosciences, CA, USA) in accordance with the manufacturer’s protocol. A BluePippin device (Sage Science, Inc., Beverly, MA, USA) was used to select 20 kb insert size fragments for the SMRTbell Template library. Quality inspection and quantification of the size-selected library were done using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit 4.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Prepared whole-genome libraries were sequenced on a PacBio Sequel sequencer (Pacific Biosciences, Menlo Park, CA, USA) with one SMRT cell at the Engineering Research Center of the Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Changchun, China. The genome was assembled using SMARTdenovo as described below, in accordance with www.github.com/smartdenovo . The completeness of the assembled genome was evaluated using the Core Eukaryotic Genes Mapping Approach (CEGMA) [19 (link)] and Benchmarking Universal Single-Copy Orthologs (BUSCO v3) [20 (link),21 (link)] with conserved orthologous gene profiles for fungi.
4
Tissue-specific RNA-seq and Isoform-sequencing
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Mixed samples from six healthy tissues (root, young leaf, mature leaf, flower bud, mature flower, and fruit) were collected for RNA extraction using the TRIzol Universal Reagent (Tiangen Biotech, Beijing, China). RNA sequencing (RNA-seq) libraries were prepared using the TruSeq Sample Preparation Kit (Illumina) and sequenced on the HiSeq X Ten platform. Clean Illumina data were obtained by removing adapters and low-quality reads from the raw data. For Isoform-sequencing, cDNA products were amplified using KAPA HiFi PCR kits (Kapa Biosystems, Wilmington, MA, USA), followed by purification using the SMRTbell Template Prep Kit (Pacific Biosciences, Menlo Park, CA, USA). Libraries were sequenced on the PacBio Sequel platform.
5
Full-length Transcriptome Generation from PacBio
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The library preparation and full-length transcriptome were performed by the Annoroad Gene Technology Corporation (Beijing, China). Briefly, equal quantities of total RNA from each sample (including D0, D12, D13, and D20) were pooled and then utilized for PacBio library preparation with the SMRTbell Template Prep Kit (Pacific Biosciences, CA, USA). The PacBio library was sequenced on the PacBio sequel machine with non-size-selected cDNAs.
Full-length transcripts were generated by the IsoSeq3 program (https://github.com/PacificBiosciences/IsoSeq3 ), including four modules of ccs, lima, cluster, and polish. Briefly, the circular consensus sequence (CCS) reads were generated from sub-reads by the ccs module, and subjected to remove primers and unwanted sequences by the lima module. Then, polyA tails and artificial concatemers were removed to generate full-length non-concatemer (FLNC) transcripts, which were clustered together by the cluster module. Finally, a consensus sequence was produced for each clustered transcript by the polish module. The high-quality full-length transcripts obtained were subsequently utilized as references for ssRNA-seq and iTRAQ analysis.
Full-length transcripts were generated by the IsoSeq3 program (
6
Genome Assembly and Annotation from Long-Read Sequencing
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Genomic DNA was extracted from cellulose-fed culture. DNA sequencing libraries were then prepared with the SMRTbell Template Prep Kit following the manufacturer’s instructions (Pacific Biosciences). Small fragments (lower than 20 kb of the SMRTbell template) were removed using the BluePippin Size Selection System for large-insert libraries. The SMRTbell library was first sequenced using 1 SMRT cell (Pacific Biosciences) and C4 chemistry (DNA sequencing Reagent Kit 4.0). A 240-min movie was then made for each SMRT cell using the PacBio RS II sequencing platform. De novo assembly was conducted by using the hierarchical genome assembly process (version 2.3) workflow and consensus polishing with Quiver (61 (link)). Reads were down-sampled to around 318× coverage to improve assembly statistics (N50). Finally, we checked the form for each contig using MUMmer (62 (link)) and trimmed the ones with self-similar ends for the closed circular genome. Putative gene CDSs were identified by Glimmer (63 (link)). Functional annotations were assigned by blasting predicted CDSs with Blastall alignment against the NCBI (National Center for Biotechnology Information) Non-Redundant Database (nr), Pfam, COG, and Prosite public databases for all species. Noncoding genes for rRNAs and tRNAs were predicted by RNAmmer (64 (link)) and tRNAscan-SE (65 (link)), respectively.
7
PacBio Iso-Seq Library Preparation
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The Iso-Seq library was prepared according to the Pacific Biosciences protocol. Briefly, 1 µg of polyA mRNA was reversely transcribed into cDNA using the Clontech SMARTer PCR cDNA Synthesis Kit. The optimal amplification cycle number was determined for generating dsDNA. After amplification, PCR product was purified using AMPure PB beads (Pacific Biosciences, Menlo Park, CA, USA) and was subjected to construction of SMRTbell library using SMRTBell Template Prep Kit (Pacific Biosciences, Menlo Park, CA, USA). The library was then sequenced on a Pacific Biosciences RSII sequencer using P2.1–C2.1 chemistry with 20 h movies (Pacific Biosciences, Menlo Park, CA, USA).
8
Generating High-Quality Genome Assemblies
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High-molecular weight (HMW) DNA fragments were separated from the extracted genomic DNA samples using BluePippin Size Selection System (Sage Science, Beverly, United States) through pulse-field gel electrophoresis and eight 20-kb sequencing libraries were constructed using SMRTbell Template Prep Kit (Pacific Biosciences, Menlo Park, United States) following the manufacturer’s instructions. The libraries (16 SMRT cells) were sequenced on the PacBio RSII platform (Pacific Biosciences). Contig sequences were assembled from the 151.99 Gb PacBio sequencing reads using Hifiasm v0.124 and polished by Racon v1.4.35 (Cheng et al., 2021 (link)). The Illumina sequencing reads were aligned to the assembled contigs using Bwa-mem v2.2.16 and the draft assembly was corrected by the aligned short sequences using Pilon v1.247 (Walker et al., 2014 (link)).
9
PacBio Isoform Sequencing of Plant Transcriptomes
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Total RNA for each of the four organs was constructed library separately according to the PacBio Isoform Sequencing (Iso-Seq) experimental protocol. Poly(A) mRNA was isolated from total RNA using Dynabeads Oligo (dT) magnetic beads (Dynal, Life Technologies, Carlsbad, CA, USA). First-strand cDNA was synthesized from poly(A) mRNA using a Clontech SMARTer PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, USA). Then, after optimization of the PCR cycles, large-scale PCR amplification was used to synthesize second-strand cDNA. Size selection was performed with a BluePippin Size Selection System (Sage Science, Beverly, MA, USA) for each sample. Unfiltered fragments and >4 kb fragments were equally mixed and used to construct the SMRTbell library with the SMRTBell Template Prep Kit (Pacific Biosciences, Menlo Park, CA, USA). SMRTbell libraries were sequenced on the PacBio Sequel platform (Pacific Biosciences, Menlo Park, CA, USA) using V2 chemistry with 10 h movies. A total of eight SMRT cells were used for sequencing.
The PacBio Iso-Seq FL transcriptome data were deposited in the Sequence Read Archive (SRA) of NCBI as follows: root: SRR11715805; stem: SRR11715804; leaf: SRR11715803; strobilus: SRR11715802.
The PacBio Iso-Seq FL transcriptome data were deposited in the Sequence Read Archive (SRA) of NCBI as follows: root: SRR11715805; stem: SRR11715804; leaf: SRR11715803; strobilus: SRR11715802.
10
Full-length 16S rRNA Gene Sequencing Protocol
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TransStart® FastPfu DNA Polymerase (TransGen Biotech, Beijing, China) was used for all PCR assays. For the full-length 16S rRNA gene sequencing, a specific primer set (27F: 5′-CCTACGGGNGGCWGCAG-3′, 1492R: 5′-GACTACHVGGGTAT CTAATCC-3′) was used according to the 16S metagenomic sequencing library preparation procedure (Pacific Biosciences, Menlo Park, CA, USA). The mixture of PCR products was separated using 2% agarose gel electrophoresis stained with SYBR green loading dye. PCR products were purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Sequencing libraries were generated using a SMRTbell Template Prep Kit (Pacific Biosciences, Menlo Park, CA, USA). The quality of the library was assessed on a Qubit 4.0 fluorometer (Thermo Scientific, Waltham, MA, USA) and Femto Pulse system (Agilent, Santa Clara, CA, USA). Finally, the library was sequenced on a PacBio Sequel platform.
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