Full-length cDNA was synthesized from the purified total RNA using the SMARTer PCR cDNA Synthesis Kit (Takara Clontech Biotech, Dalian, China) following the manufacturer’s protocol, and large-scale PCR was conducted to produce more double-stranded cDNA templates. Size selection was then performed to generate SMRTbell™ libraries using a PacBio Template Prep Kit (PacBio, Menlo Park, CA, USA). Subsequently, full-length transcriptome sequencing of Z. nitidum was performed using the Pacific Sequel platform.
Pacbio template prep kit
Manufactured by Pacific Biosciences
Sourced in United States
The PacBio Template Prep Kit is a laboratory equipment product designed to prepare DNA samples for sequencing on PacBio sequencing platforms. The kit provides the necessary reagents and materials for DNA sample preparation, including steps such as DNA extraction, fragmentation, and adapter ligation.
Automatically generated - may contain errors
Lab products found in correlation
Idt barcoded primers, by Integrated DNA Technologies (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Bluepippin size selection system, by Sage Science (2 mentions)
Experion electrophoresis system, by Bio-Rad (1 mentions)
Bluepippin system, by Pacific Biosciences (1 mentions)
Pacbio rs 2 system, by Pacific Biosciences (1 mentions)
Rna seq data, by Illumina (1 mentions)
Pacbio sequel sequencer, by Novogene (1 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
Agilent 2100 bioanalyzer, by Thermo Fisher Scientific (1 mentions)
Smarter pcr cdna synthesis kit, by Takara Bio (1 mentions)
Ampure pb bead, by Pacific Biosciences (1 mentions)
G tube device, by Covaris (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
8 protocols using pacbio template prep kit
1
Full-length Transcriptome Sequencing of Z. nitidum
2
PacBio Full-length cDNA Iso-Seq Analysis
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Full-length cDNA Iso-Seq template libraries for PacBio Iso-Seq analysis were constructed and sequenced at the DNA Technologies & Expression Analysis Core Facility of the UCD Genome Center. FL double-stranded cDNA was generated from total RNA (2 µg per tissue) using the Lexogen TeloTM prime Full-length cDNA Kit (Lexogen, Inc., Greenland, NH). Tissue-specific cDNAs were first barcoded by PCR (16–19 cycles) using IDT barcoded primers (Integrated DNA Technologies, Inc., Coralville, IA), and then bead-size selected with AMPure PB beads (2 different size fractions of 1X and 0.4X). The 9 cDNAs were pooled in equimolar ratios and used to prepare a SMRTbell™ library using the PacBio Template Prep Kit (PacBio, Menlo Park, CA). The SMRTbell™ library was then sequenced across 4 Sequel v2 SMRT cells with polymerase 2.1 and chemistry 2.1 (P2.1C2.1).
PacBio raw reads were processed using the Iso-Seq3 v.3.0 workflow following PacBio recommendations [69 ]. CCSs were generated using the program “ccs.” The CCSs were demultiplexed and cleaned of cDNA primers using the program “lima.” Afterward, CCS clustering and polishing was performed using the program “Iso-Seq3,” to generate HQ FL sequences for each of the 9 tissues. FLnc and HQ clusters were aligned onto the new “Chandler” assembly v2.0 with minimap2 v.2.12-r827, including the parameter “-ax splice” [70 (link)].
PacBio raw reads were processed using the Iso-Seq3 v.3.0 workflow following PacBio recommendations [69 ]. CCSs were generated using the program “ccs.” The CCSs were demultiplexed and cleaned of cDNA primers using the program “lima.” Afterward, CCS clustering and polishing was performed using the program “Iso-Seq3,” to generate HQ FL sequences for each of the 9 tissues. FLnc and HQ clusters were aligned onto the new “Chandler” assembly v2.0 with minimap2 v.2.12-r827, including the parameter “-ax splice” [70 (link)].
3
Giant Sequoia Transcriptome Profiling
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RNA was isolated from giant sequoia roots, foliage, and cambium using a LiCl-Urea buffer followed by cleanup using Zymo columns and reagents (Zymo Research, Irvine, CA). RNA quality was assessed using an Experion Electrophoresis System (Bio-Rad, Hercules, CA) and Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA). Double-stranded cDNA was generated from total RNA (2 µg per tissue) using the Lexogen TeloTM prime Full-length cDNA Kit (Lexogen, Inc., Greenland, NH, USA).
Tissue-specific cDNAs were first barcoded by PCR (16-19 cycles) using IDT barcoded primers (Integrated DNA Technologies, Inc., Coralville, Iowa), and then bead-size selected with AMPure PB beads (two different size fractions of 1X and 0.4X). The three cDNAs were pooled in equimolar ratios and used to prepare a SMRTbell™ library using the PacBio Template Prep Kit (PacBio, Menlo Park, CA). The SMRTbell™ library was then sequenced on a Sequel v2 SMRT cell with polymerase 2.1 and chemistry 2.1 (P2.1C2.1) on one PacBio Sequel v2 SMRT cell at the UC Davis Genome Center DNA Technologies Core Facility.
Tissue-specific cDNAs were first barcoded by PCR (16-19 cycles) using IDT barcoded primers (Integrated DNA Technologies, Inc., Coralville, Iowa), and then bead-size selected with AMPure PB beads (two different size fractions of 1X and 0.4X). The three cDNAs were pooled in equimolar ratios and used to prepare a SMRTbell™ library using the PacBio Template Prep Kit (PacBio, Menlo Park, CA). The SMRTbell™ library was then sequenced on a Sequel v2 SMRT cell with polymerase 2.1 and chemistry 2.1 (P2.1C2.1) on one PacBio Sequel v2 SMRT cell at the UC Davis Genome Center DNA Technologies Core Facility.
4
High-quality reference genome assembly
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To obtain a representative, high-quality reference genome for short-read mapping, S. Concord isolate ITM_8091960 was sequenced using the PacBio RSII system (Pacific Biosciences, California, USA). This isolate originated from a stool sample of an Ethiopian child that presented at the travel clinic of ITM Antwerp in 2008. DNA was prepared using the PacBio Template Prep Kit (Pacific Biosciences, California, USA) and the BluePippin™ system for size selection, for sequencing with the PacBio RSII system at the Wellcome Sanger Institute (Hinxton, UK). Reads were assembled de novo using the HGAP56 (link) protocol v3.0 implemented in smrtanalysis v2.3.0, resulting in one chromosome-sized contig, and five small contigs. Circlator57 (link) v1.5.5 was used to remove self-compatible ends and rearrange the start positions of the contigs at the dnaA gene, or a predicted gene. The resulting contigs were polished twice using Quiver56 (link).
5
Full-length Transcriptome Sequencing of C. deserticola and H. ammodendron
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Full-length transcriptomes of C. deserticola and non-parasitized H. ammodendron were also obtained by third-generation sequencing. The isoform sequencing library was prepared according to the Iso-SeqTM protocol as described by Pacific Biosciences. Briefly, 0.5 μg of poly(A) RNA was reverse transcribed into cDNA using Clontech SMARTerTM cDNA Synthesis Kit (Takara, Japan). BluePippin system was used for size selection of cDNAs, and SMRTbellTM libraries were constructed via PacBio Template Prep Kit (Pacific Biosciences, USA). The libraries were sequenced on a PacBio Sequel sequencer by Novogene company (Beijing, China). Sequence data were processed via SMRTlink 5.1 software.51 (link) Circular consensus sequence (CCS) was generated from subread BAM files. CCS.BAM files were retrieved and classified into full-length and non-full-length reads via isoform-level clustering (ICE). Additional nucleotide errors in consensus reads were corrected using the Illumina RNA-seq data with the LoRDEC software.52 (link) Any redundancy in consensus reads was removed by CD-hit to obtain final transcripts of C. deserticola and non-parasitized H. ammodendron (abbreviated as CD_FL and HA_FL hereafter) for the subsequent analysis.56 (link)
6
Comprehensive Full-Length Transcriptome Analysis
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To obtain the comprehensive full-length transcriptome, equal amount of the RNA samples from the seven time points were pooled and used for Iso-seq library preparation. First stand cDNA synthesis was performed using the SMARTer PCR cDNA Synthesis Kit (Clontech) and subsequently used for large-scale PCR to generate double-stranded cDNA by PrimeSTAR GXL DNA Polymerase (Clontech) according to the manufacturers’ instructions. The amplified cDNA was then cleaned up by Ampure PB Beads, and full-length transcripts with sizes up to 4 kb were collected. At the same time, < 4 kb transcripts were collected using the BluePippin Size-Selection System (Sage Science, Inc., MA, USA) to enrich the transcript concentration. Both < 4 kb and > 4 kb transcripts were separately used for SMRTbell library construction using the PacBio Template Prep Kit (Pacific Biosciences of California, Inc., California, USA). The established libraries were validated using the Agilent 2100 Bioanalyzer and quantified using the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Finally, two SMRT cells were run on the PacBio Sequel System using P6-C4 chemistry.
7
PacBio SMRT Sequencing Protocol
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The PacBio SMRT sequencing was started using the PacBio Template Prep Kit (Pacific Biosciences, Cat NO,101–357-000) to generate SMRTbell libraries, which was created by ligating hairpin adapters to double-stranded DNA, thereby circularizing them into a construct termed a SMRT bell. Then, the SMRTbell libraries were sequenced with the Pacific Sequel platform (Pacific Biosciences, Cat NO, 101–310-500). A primer and a polymerase were annealed to the adapter and loaded into SMRT Cell. Each SMRT Cell contained millions of nanoscale observation chambers called Zero Mode Wave guides, where a single DNA polymerase attached to a single DNA template was anchored to the bottom and the polymerase continuously incorporated fluorescently labeled nucleotides to emit fluorescence signals, while the camera recorded in real time to realize simultaneous synthesis and sequencing.
8
PacBio Sequel Long-Read Sequencing
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Qualified DNA was sheared using Covaris g-TUBE device. The fragmented DNA was repaired using PacBio Template Prep Kit (Pacific Biosciences, United States). The enrichment for long fragments was done by BluePippin size selection system (Sage Science) to construct a 20 kb library. After DNA purification using AMPure® PB beads (Pacific Biosciences, United States), the DNA fragments were ligated to the hairpins (SMRTbellTM templates). The library quality was checked by Agilent Bioanalyzer 2100 (Agilent Technologies, CA, United States) and Qubit 2.0 Fluorometer (Invitrogen, Life Technologies, CA, United States). The prepared SMRTbellTM templates was bound with magbead and loaded on a SMRT cell of PacBio Sequel platform. Single-molecule real-time (SMRT®) DNA sequencing (Berlin et al., 2015 (link)) was performed in Nextomics Biosciences Co., Ltd (Wuhan, China) according to the manufacturer’s protocol (Pacific Biosciences, CA, United States). Raw reads were processed by the SMRT Link v2.3.0 in the default mode to remove the adaptor sequences and low quality reads (below quality 0.8), and the filtered reads (6.3 G) were assembled to contigs with no gaps by CANU with default parameters (Koren et al., 2017 (link)). The sequencing data generated were deposited in NCBI Short Read Archive database (SRA accession: PRJNA649095 ).
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