Total RNA of the 24 samples was mixed together in equal parts (0.5 μg for each sample). Poly(A) RNA was isolated using the Poly(A) Purist TM Kit (Ambion Inc., Grand Island, NY, USA). RNA was reverse transcribed into cDNA using the SMARTer™ PCR cDNA Synthesis Kit (Clontech Laboratories Inc., Mountain View, CA, USA). We used the BluePippin size selection system (Sage Science, Beverly, MA, USA) to generate cDNA fractions with different sizes, including 1–2 kb, 2–3 kb, and 3–6 kb. These libraries were then constructed with the Pacific Biosciences’ SMRTbell Template Prep Kit 2.0 (Pacific Biosciences, https://www.pacb.com , accessed on 1 August 2019) according to the manufacturer’s protocol. A total of five SMRT cells (two SMRT cells for libraries of 1–2 kb, two SMRT cells for libraries of 2–3 kb, and one SMRT cell for libraries of 3–6 kb) were sequenced on the PacBio RS II platform. The raw sequencing reads from the PacBio RSII SMRT cells were processed through the SMRT-Portal analysis suite to subread sequences for further processing.
Smrtbell template prep kit 2
Manufactured by Pacific Biosciences
Sourced in United States
The SMRTbell Template Prep Kit 2.0 is a laboratory equipment product from Pacific Biosciences. It is designed for the preparation of DNA samples for sequencing using Pacific Biosciences' SMRT (Single Molecule, Real-Time) sequencing technology.
Automatically generated - may contain errors
Lab products found in correlation
Bluepippin size selection system, by Sage Science (2 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (2 mentions)
Pacbio sequel 1, by Pacific Biosciences (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Smarter pcr cdna synthesis kit, by Takara Bio (1 mentions)
Poly a purist tm kit, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 reagent kit v2, by Illumina (1 mentions)
Short read, by Illumina (1 mentions)
Sequel sequencing kit v2, by Pacific Biosciences (1 mentions)
Qiaseq fx dna library kit, by Qiagen (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Pacbio sequel, by Pacific Biosciences (1 mentions)
Chromium single cell 3 reagent kit, by Illumina (1 mentions)
Chromium single cell 3 v3reagent kits, by Genomics Plc (1 mentions)
Qubit hs, by Thermo Fisher Scientific (1 mentions)
Pacbio sequel 2 platform, by Pacific Biosciences (1 mentions)
Matrix e, by MP Biomedicals (1 mentions)
Sequel binding kit 3, by Pacific Biosciences (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Smrt sequencing technology, by Pacific Biosciences (1 mentions)
10 protocols using smrtbell template prep kit 2
1
Long-read sequencing of total RNA
2
Csp_BJ Genome Sequencing and Annotation
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The genomic DNA library of Csp_BJ was constructed by using a QIAseq FX DNA Library Kit (Qiagen) according to the manufacturer’s instructions, and then by paired-end sequencing using an Illumina NextSeq 500 platform with a 300-cycle NextSeq 500 reagent kit v2 (Illumina). The metagenomic samples were sequenced by single-end sequencing by using a 150-cycle NextSeq 500 reagent kit v2 (Illumina). The complete genome sequence of the strain was determined by using a PacBio Sequel (Pacific BioSciences) sequencer with a Sequel SMRT Cell 1M v2 (four/tray) and a Sequel sequencing kit v2.1 (Pacific BioSciences) for long-read sequencing (insert size, ≈10 kb). High quality genomic DNA was used to prepare a SMRTbell library by using a SMRTbell Template Prep Kit 2.0 (Pacific Biosciences). The draft genome contigs were assembled by using A5-Miseq software with Illumina short reads [33 (link)]. The circular genome sequence was constructed by using Canu version 1.4 [34 (link)], Minimap version 0.2-r124 [35 (link)], racon version 1.1.0 [36 (link)], and Circlator version 1.5.3 [37 (link)] with long read data. Error correction of the circular sequence was performed by using Pilon version 1.18 with short reads [38 (link)]. Annotation was performed in DFAST version 1.0.8 [39 (link)] and NCBI-BLASTP/BLASTX against deposited Chromobacterium complete genome sequences.
3
Single-cell multiome analysis of mouse organ of Corti
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Single-cell capture and cDNA amplification of postnatal day (P)7 mice organ of Corti were performed using Chromium Single Cell 3’ V3Reagent Kits (10× Genomics, USA) and Gel Beads with the following modifications. In brief, the RT time was increased to 2 h to potentially increase the efficacy of reverse transcription of longer transcripts. Finally, the amplified cDNAs were split into two pools, one pool was used for Illumina 3′ sequencing for differential gene expression analysis, and the other pool was used for PacBio long-read sequencing to detect full-length RNA isoforms.
Illumina library preparation was performed using 100 ng of amplified cDNA following the Chromium Single Cell 3’ Reagent Kits V3 User Guide. The final libraries were sequenced on the Illumina NovaSeq 6000 platform (Illumina, USA). Pacbio library preparation was performed using the SMRTbell™ Template Prep Kit 2.0 (Pacific Biosciences, USA), and then assessed using an Agilent Bioanalyzer 2100 system (Agilent Technologies, USA) and Qubit HS (Life Technologies, USA) before sequencing. SMARTbell sequencing libraries were bound to polymerases using the Sequel Binding Kit 2.1 and V3 Primers. The polymerase template complexes were bound to MagBeads with the PacBio MagBio Binding Kit. Sequencing reactions were performed using the PacBio Sequel II platform (Pacific Biosciences, USA) in CCS mode.
Illumina library preparation was performed using 100 ng of amplified cDNA following the Chromium Single Cell 3’ Reagent Kits V3 User Guide. The final libraries were sequenced on the Illumina NovaSeq 6000 platform (Illumina, USA). Pacbio library preparation was performed using the SMRTbell™ Template Prep Kit 2.0 (Pacific Biosciences, USA), and then assessed using an Agilent Bioanalyzer 2100 system (Agilent Technologies, USA) and Qubit HS (Life Technologies, USA) before sequencing. SMARTbell sequencing libraries were bound to polymerases using the Sequel Binding Kit 2.1 and V3 Primers. The polymerase template complexes were bound to MagBeads with the PacBio MagBio Binding Kit. Sequencing reactions were performed using the PacBio Sequel II platform (Pacific Biosciences, USA) in CCS mode.
4
PacBio Metagenomic DNA Sequencing
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Bacterial pellets were resuspended in 200 µL of 1× PBS and transferred to a 2-mL bead beating tube (Matrix E; MP Biomedicals). Proteinase K (20 µL; Qiagen) was added, and the cells homogenized (SPEX 1600 MiniG; 1 min; 1,500 Hz Fisher Scientific). DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit according to the manufacturer’s instructions (Qiagen). The DNA was quantified using the 1× dsDNA HS kit (ThermoFisher Scientific on a Qubit). DNA was prepped with the SMRTbell Template Prep Kit 2.0 (Pacific Biosciences) to make PacBio SMRTbell libraries with barcodes sourced from the Barcoded Overhang Adaptor Kit 8A and 8B (Pacific Biosciences). The sequencing primers were annealed and bound to Polymerase 3.0 using the Sequel Binding Kit 3.0 (Pacific Biosciences). The bound complex was purified and sequenced on a PacBio Sequel I using an SMRT Cell M1 v3 tray (Pacific Biosciences). The spike-in controls for each PacBio Sequel I run were from the Internal Control Kit 3.0 (Pacific Biosciences).
5
Long-read Transcriptome Sequencing
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The total RNA of each sample was pooled together in equimolar ratios, and approximately 2 μg of the total RNA was subsequently used for the FL cDNA synthesis by a SMARTer™ PCR cDNA Synthesis Kit (Takara Clontech Biotech, Dalian, China) according to the standard protocol. Size fractionation and selection (1–2 kb, 2–3 kb, 3–6 kb and 5–10 kb) of the FL cDNA was carried out using the BluePippin™ Size Selection System (Sage Science, Beverly, MA), and a re-amplification was than conducted for the selected FL cDNA fragments with PCR. The three SMRTbell Template libraries (1–2 kb, 2–3 kb and >3 kb) were constructed with SMRTBell Template Prep Kit 2.0 (Pacific Biosciences, USA) following the manufacturer’s recommendations. The library quantification was performed using Qubit System,21 and the size range of the library was detected with Agilent Bioanalyzer 2100 system (Agilent Technologies, USA). The single-molecule real-time (SMRT) cells for the three libraries were then sequenced on the Pacific Bioscience RS II platform using P6-C4 reagent with 4 h sequencing movies (Pacific Biosciences, Menlo Park, CA, USA).
6
Long-read Genomic DNA Sequencing
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Genomic DNA was isolated from peripheral blood according to standard procedures and subjected to long-read genome Hi-Fi sequencing using the SMRT sequencing technology (Pacific Biosciences, Menlo Park, CA, USA). Library preparation was performed using the SMRTbell™ Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA) following the manufacturer’s instructions. Size selection was performed using a BluePippin DNA size selection system (target fragments ~ 15–18 kb). Sequence primer V2 and polymerase 2.0 were used for binding. Subsequently, the SMRTbell library was loaded on an 8 M SMRTcell and sequencing was performed on a Sequel II system (Pacific Biosciences, Menlo Park, CA, USA). Circular consensus sequencing (CCS), Hi-Fi reads, were generated using the CCS (v4.2.0) tool and were aligned to the GRCh37/hg19 reference genome with pbmm2 (v.1.3.0). The unique molecular yield was 93.46 Gb and the post-alignment Hi-Fi- coverage was 12× [Mosdepth v0.3.1, (Pedersen and Quinlan 2018 (link))]. SV calling was performed using PBSV (v2.4.0) and annotation was applied using an in-house SV annotation pipeline.
7
Full-length Transcriptome Profiling using PacBio Sequencing
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Colo320 cDNA was prepared using the SMARTer cDNA synthesis kit (Clonetech) and converted into a SMRTbell library using the SMRTbell Template Prep Kit 2.0 (Pacific Biosciences) according to the manufacturer's instructions. The samples were sequenced on a PacBio Sequel I (Pacific Biosciences). Obtained data were analyzed using the Iso-Seq3 in the PacBio SMRT Analysis (v. 6.0) as described elsewhere (25) . Briefly, circular consensus sequences with both 5 0 and 3 0 primers were regarded as full-length reads, and they were pooled for isoform-level clustering analysis. The Arrow algorithm in the SMRT Link software called high-quality sequences (predicted consensus accuracy ≥ 99%), which were then mapped to GRCh38.p13 using minimap2 (v. 2.1). The full-length isoforms were ultimately annotated and characterized using a SQANTI2 pipeline (26) . Library preparation and sequencing were performed by Macrogen (Korea).
8
Whole-Genome Sequencing of Asian Moon Scallop
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The Asian moon scallops (A. pleuronectes) were gathered from the Tung Ping Chau Bay (near Tung Chung village, Guangzhou) for whole-genome sequencing. The DNA samples were stored at the key laboratory of marine genetics and breeding, Ministry of Education, Ocean University of China (specimen code: OUC-MGB-2019-Apl-08). Dissected tissues were immediately frozen in liquid nitrogen and preserved at −80 °C for DNA extraction. The conventional hexadecyltrimethylammonium bromide (CTAB) procedure was used to extract high molecular-weight genomic DNA (gDNA) from the adductor muscle [43] . The pair-end libraries with an insert size of 350 bp were built from gDNA by utilizing Illumina genomic DNA sample preparation kits (Catalog No. 20060060, Illumina, San Diego, CA), following the manufacturer’s standard protocols, then sequenced on the Illumina Xten platform. A long-read DNA library for gDNA derived from the same individual was constructed using SMRTbell template prep kit 2.0 (Catalog No. 101-685-400, PacBio, Menlo Park, CA) on the Pacific Biosciences (PacBio) Sequel single-molecule real-time (SMRT) platform.
9
Genome and Transcriptome Sequencing of P. zhonghuajia
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Colonies of P. zhonghuajia were reared on mature larvae of Sitotroga cerealella (Oliver) (Lepidoptera: Gelechiidae) with wheat bran in a climate chamber at 25 ± 1 °C with 60 ± 5% relative humidity (RH) at Changli Institute of Pomology, Hebei Academy of Agriculture and Forestry Sciences. There were 2000 seven-day-pregnant mites used for Illumina whole-genome and PacBio sequencing, respectively. Genomic DNA was extracted using the QIAGEN DNeasy Blood & Tissue kit, which was then used to construct a 350 bp insert-size library using the Truseq DNA PCR-free kit for sequencing on the Illumina NovaSeq 6000 platform and a 15 kb insert-size library using the SMRTbell™ Template Prep Kit 2.0 for sequencing on the PacBio Sequel II platform. The whole-individual transcriptome was performed using RNA-Seq from 2000 one-day-old mites and 2000 seven-day-pregnant mites with three biological replicates for each group. Total RNA was extracted using the TRIzol™ Reagent kit and the RNA-Seq library was constructed using TruSeq RNA v2 kit. DNA/RNA extraction, library construction, and sequencing were performed at Berry Genomic (Beijing, China).
10
Hybrid Genome Assembly of Microbial Isolates
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The extracted genomic DNA of 68 isolated strains was sheared to yield DNA fragments. The genome sequences were determined by the whole-genome shotgun strategy using PacBio Sequel and Illumina MiSeq sequencers. The library of the Illumina Miseq 2 x 300bp paired-end sequencing was prepared using TruSeq DNA PCR-Free kit (target length = 550bp) and all the MiSeq reads were trimmed and filtered with a >20 quality value (QV) using FASTX-toolkit (hannonlab.cshl.edu/fastx_toolkit). The library of the PacBio Sequel sequencing was prepared using SMRTbell template prep kit 2.0 (target length = 10 -15kbp) without DNA shearing. After removal of internal control and adaptor trimming by Sequel, the error correction of the trimmed reads was performed using Canu (v1.8) with additional options (corOutCoverage = 10,000, corMinCoverage = 0, corMhapSensitivity = high). De novo hybrid assembly of the filter-passed MiSeq reads and the corrected Sequel reads were performed using Unicycler (v0.4.8), which contained checks for overlapping and circularization to generate circular contigs. The gene prediction and annotation of the generated contigs were performed using the Rapid Annotations based on Subsystem Technology (RAST) server 56 and Prokka software tool 57 . Default parameters were used unless otherwise specified.
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