Bluepippin size selection

Manufactured by Sage Science

Sourced in United States

The BluePippin is a size selection instrument that allows for the precise isolation of DNA fragments within a specified size range. It provides a reliable and automated method for the purification of DNA samples, ensuring the recovery of targeted molecular weights.

Automatically generated - may contain errors

Lab products found in correlation

Ampure xp bead, by Beckman Coulter (4 mentions) G tube device, by Covaris (2 mentions) Dna polymerase binding kit p6, by Pacific Biosciences (2 mentions) G tube, by Covaris (2 mentions) Pacbio rs 2, by Pacific Biosciences (2 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions) Clontech smarter pcr cdna synthesis kit, by Takara Bio (1 mentions) Rsii platform, by Pacific Biosciences (1 mentions) Tapestation 4200, by Agilent Technologies (1 mentions) Nextseq 550 system, by Illumina (1 mentions) Spectramax quant dsdna assay kit, by Molecular Devices (1 mentions) Gemini xps fluorometer, by Molecular Devices (1 mentions) Biomek fxp automated workstation, by Beckman Coulter (1 mentions) Smrtbell template preparation kit, by Pacific Biosciences (1 mentions) D1000 screentape, by Agilent Technologies (1 mentions) G tube fragmentation, by Covaris (1 mentions) Paired end kits, by Illumina (1 mentions) Dneasy blood tissue kit, by Qiagen (1 mentions) Hiseq 2500, by Illumina (1 mentions) Calculator in rs remote, by Pacific Biosciences (1 mentions)

8 protocols using bluepippin size selection

PacBio ISO-Seq Workflow: cDNA Synthesis and Sequencing

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The library was prepared according to the PacBio ISO-Seq experimental workflow (Supplementary Figure S1). The first cDNA strand was synthesized from purified polyA RNAs using a Clontech SMARTer PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, United States). After PCR optimization, large-scale PCR was performed to synthesize second strand cDNA for BluePippin size selection (Sage Science, Inc., Beverly, MA, United States) with size ranges of 0-1 kb, 1-2 kb, 2-3 kb, and 3-6 kb. After size selection, another amplification was performed, and amplified, size selected cDNA products were made into SMRTbell template libraries (0-1 kb, 1-2 kb, 2-3 kb, and 3-6 kb) according to the manufacture’s instruction.
Libraries were prepared by annealing a sequencing primer (SMRTbell Template Prep Kit 1.0) and binding polymerase to the primer-annealed template. Sequencing was performed on a PacBio RS II platform. A total of seven SMRT cells were conducted in this study (Supplementary Table S1).

Xu Q., Zhu J., Zhao S., Hou Y., Li F., Tai Y., Wan X, & Wei C. (2017). Transcriptome Profiling Using Single-Molecule Direct RNA Sequencing Approach for In-depth Understanding of Genes in Secondary Metabolism Pathways of Camellia sinensis. Frontiers in Plant Science, 8, 1205.

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Bacterial DNA Isolation and Sequencing

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DNA was extracted from bacterial isolates using the Agencourt GenFind DNA Isolation Kit and the Biomek FX^P Automated Workstation (Beckman Coulter). A short-read NGS library was prepared using the Nextera DNA Flex or XT Library Prep Kit (Illumina, San Diego, CA, USA). DNA quantification was performed in a microplate using the SpectraMax Quant dsDNA Assay Kit and the Gemini XPS Fluorometer (Molecular Devices, San Jose, CA, USA), or in a single tube using the Qubit 2.0 fluorometer (Thermo Fisher Scientific, Springfield Township, NJ, USA). Library quality was examined using the 4200 TapeStation and D1000 ScreenTape (Agilent, Santa Clara, CA, USA). Paired-end sequencing (2 × 150 cycles) was performed using the NextSeq 550 system (Illumina). The Pacific Biosciences (PacBio, Menlo Park, CA, USA) RSII single-molecule real-time (SMRT) sequencing system was employed for the long-read sequencing of A. baumannii PB364. The long-read NGS library was processed using g-TUBE fragmentation (Covaris, Woburn, MA, USA), BluePippin size selection (Sage Science, Beverly, MA, USA) and a SMRTbell template preparation kit (PacBio). Mainly, the manufacturers’ standard protocols were followed.

Huang W., Wang G., Yin C., Chen D., Dhand A., Chanza M., Dimitrova N, & Fallon J.T. (2019). Optimizing a Whole-Genome Sequencing Data Processing Pipeline for Precision Surveillance of Health Care-Associated Infections. Microorganisms, 7(10), 388.

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Long-Read PacBio and Short-Read Illumina Sequencing of Gayal Genome

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Genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen) and sheared using a g-TUBE device (Covaris) with 20 kb settings. Sheared DNA was purified and concentrated with AmpureXP beads (Agencourt) and used for SMRT bell preparation according to the manufacturer’s protocol [Pacific Biosciences; 20-kb template preparation using BluePippin size selection (Sagescience)]. Size-selected and isolated SMRT bell fractions were purified using AmpureXP beads (Beckman Coulter, Inc.). Finally, purified SMRT bells were used for primer (V3)-and polymerase (2.0) binding according to the manufacturer’s binding calculator (Pacific Biosciences). Single-molecule sequencing was performed on a PacBio Sequel system yielding a total of 16,978,079 filtered subreads with average lengths of 8,912 bp for gayal. Finally, only PacBio subreads ≥500 bp were used for genome assembly. Five 270 bp paired-end libraries were constructed using Illumina’s paired-end kits according to the manufacturer’s instructions. The libraries were sequenced on Illumina HiSeq 2500 platforms, and 195.34 Gb short read (150 bp) data were generated. Raw reads were filtered by the removal of sequencing adaptors and contaminated reads.

Li Y., Wang S., Zhang Z., Luo J., Lin G.L., Deng W.D., Guo Z., Han F.M., Wang L.L., Li J., Wu S.F., Liu H.Q., He S., Murphy R.W., Zhang Z.J., Cooper D.N., Wu D.D, & Zhang Y.P. (2023). Large-Scale Chromosomal Changes Lead to Genome-Level Expression Alterations, Environmental Adaptation, and Speciation in the Gayal (Bos frontalis). Molecular Biology and Evolution, 40(1), msad006.

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Preparation of Long-Read SMRTbell Libraries

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The SMRTbell™ template library was prepared according to the instructions from Pacific Biosciences (Menlo Park, CA, USA) following the Procedure & Checklist—Greater Than 10 kb Template Preparation. To prepare 15 kb libraries, 8 µg genomic DNA was sheared using g-tubes™ from Covaris (Woburn, MA, USA). DNA was end-repaired and ligated overnight to hairpin adapters applying components from the DNA/Polymerase Binding Kit P6 (Pacific Biosciences). BluePippin™ Size-Selection (Sage Science, Beverly, MA, USA) to greater than 4 kb was performed according to the manufacturer’s instructions. Conditions for annealing of sequencing primers and binding of polymerase to purified SMRTbell™ template were assessed with the Calculator in RS Remote (Pacific Biosciences). SMRT sequencing was carried out on the PacBio RSII (Pacific Biosciences) taking one 240-min movie for one to three SMRT cells per isolate using P6 Chemistry.

Ramli S.R., Bunk B., Spröer C., Geffers R., Jarek M., Bhuju S., Goris M., Mustakim S, & Pessler F. (2021). Complete Genome Sequencing of Leptospira interrogans Isolates from Malaysia Reveals Massive Genome Rearrangement but High Conservation of Virulence-Associated Genes. Pathogens, 10(9), 1198.

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Long-read genome sequencing protocol

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SMRTbell template libraries were prepared according to the instructions from Pacific Biosciences, following the Procedure & Checklist—Greater Than 10 kb Template Preparation document. Briefly, for the preparation of 15 kb libraries, 8 µg genomic DNA was sheared using g‐tubes from Covaris according to the manufacturer's instructions. DNA was end‐repaired and ligated overnight to hairpin adapters applying components from the DNA/Polymerase Binding Kit P6 from Pacific Biosciences. Reactions were carried out according to the instructions of the manufacturer. BluePippin Size‐Selection to greater than 4 kb was performed according to the manufacturer's instructions (Sage Science). Conditions for annealing of the sequencing primers and binding of polymerase to purified SMRTbell template were assessed with the Calculator in RS Remote (Pacific Biosciences). Single‐molecule real‐time (SMRT) sequencing was carried out on the PacBio RSII (Pacific Biosciences) taking one 240‐min movie on one SMRT cell per sample using the P6 Chemistry.

Kuzmanović N., diCenzo G.C., Bunk B., Spröer C., Frühling A., Neumann‐Schaal M., Overmann J, & Smalla K. (2023). Genomics of the “tumorigenes” clade of the family Rhizobiaceae and description of Rhizobium rhododendri sp. nov. MicrobiologyOpen, 12(2), e1352.

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SMRT Sequencing and Assembly of Brassicaceae S-locus

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We conducted SMRT sequencing (Pacific Biosciences of California, CA, USA) of two different Brassicaceae S-locus sequences in two BAC clones at the Uppsala Genome Center, National Genomics Infrastructure Sweden. DNA fragments over 10 kbp were selected using BluePippin Size selection (Sage Science, MA, USA) and the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences of California, CA, USA) was used for library preparation, with an insert size of 500 bp to 20 kb. SMRT sequencing was done on the RSII system, using P5-C3 chemistry.
To assess sequencing and assembly errors, we generated two independent libraries of one BAC clone (CgrS-BAC1), which was then subjected to independent SMRT sequencing and assembly, whereas the second BAC, (CgrS-BAC2), was sequenced once with SMRT sequencing. To assess indel errors, we also generated short-read sequencing (MiSeq, Illumina, Inc., San Diego, USA) data for the second S-locus BAC (CgrS-BAC2). The sequencing library (TruSeq PCRfree DNA sample preparation kit, Illumina, Inc., CA, USA) was prepared from 1 μg of DNA, following the manufacturers’ guidelines. We generated 1.1 million paired-end 250 bp reads on the MiSeq using v2 sequencing chemistry (Illumina, Inc., CA, USA).

Bachmann J.A., Tedder A., Laenen B., Steige K.A, & Slotte T. (2018). Targeted Long-Read Sequencing of a Locus Under Long-Term Balancing Selection in Capsella. G3: Genes|Genomes|Genetics, 8(4), 1327-1333.

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Long-read Sequencing of Nautilus Genome

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Genomic DNA was sheared by means of a g-TUBE device (Covaris) with 20-kilobase (kb) settings. Sheared DNA was purified and concentrated with AMPure XP Beads (Agencourt) for further use in single-molecule real-time (SMRT) bell preparation according to the manufacturer’s protocol (Pacific Biosciences). The 20-kb template preparation was done by BluePippin size selection (Sage Science). Size-selected and isolated SMRT bell fractions were purified with AMPure XP Beads. Finally, these purified SMRT bells were used for primer and polymerase (P6) binding according to the manufacturer’s binding calculator (Pacific Biosciences). Single-molecule sequencing was done on a PacBio RS II platform with C4 chemistry. Only PacBio subreads equal to or longer than 500 bp were used to perform N. pompilius genome assembly.

Zhang Y., Mao F., Mu H., Huang M., Bao Y., Wang L., Wong N.K., Xiao S., Dai H., Xiang Z., Ma M., Xiong Y., Zhang Z., Zhang L., Song X., Wang F., Mu X., Li J., Ma H., Zhang Y., Zheng H., Simakov O, & Yu Z. (2021). The genome of Nautilus pompilius illuminates eye evolution and biomineralization. Nature Ecology & Evolution, 5(7), 927-938.

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Long-Read Oyster Genome Assembly

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Genomic DNA was sheared by a g-TUBE device (Catalog No. 520079, Covaris, MA) with 20 kb settings. Sheared DNA was then purified and concentrated with AMPure XP beads (Catalog No. 10136224, Beckman Coulter, CA) and further used for single-molecule real-time (SMRT) bell preparation according to the manufacturer’s protocol (Pacific Biosciences, CA) and 20 kb template preparation by using BluePippin size selection (Sage Science). Size-selected and isolated SMRT bell fractions were purified with AMPure XP beads. Finally, these purified SMRT bells were used for primer and polymerase (P6) binding, according to the manufacturer’s binding calculator (Pacific Biosciences). Single-molecule sequencing was performed on a PacBio RS-II platform with C4 chemistry. Only PacBio subreads ≥ 500 bp were included for performing oyster genome assembly.

Zhang Y., Mao F., Xiao S., Yu H., Xiang Z., Xu F., Li J., Wang L., Xiong Y., Chen M., Bao Y., Deng Y., Huo Q., Zhang L., Liu W., Li X., Ma H., Zhang Y., Mu X., Liu M., Zheng H., Wong N.K, & Yu Z. (2022). Comparative Genomics Reveals Evolutionary Drivers of Sessile Life and Left-right Shell Asymmetry in Bivalves. Genomics, Proteomics & Bioinformatics, 20(6), 1078-1091.

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