Total RNA from three replicates of chicken embryos stage HH27 was isolated as previously described. This was followed by one round of poly(A) purification using oligo d(T) magnetic beads (New England Biolabs). A total of 1.5–2 µg of mRNA was fragmented to 100–150 nt using RNA Fragmentation Reagent (Thermo Fisher Scientific), followed by overnight ethanol precipitation. After centrifugation and washing, the pellets were resuspended in 10 µL H2O. An amount of 9 μL of the solution was used for the IP, and 1 µL for preparing the input libraries. The fragmented RNA was mixed with Protein G Magnetic Beads prebound monoclonal anti-m6A antibody (1 µL) from the EpiMark N6-Methyladenosine Enrichment Kit (New England Biolabs), resuspended in 300 µL EpiMark IP buffer supplemented with murine RNase inhibitor (New England Biolabs). All of the following steps were as described by the manufacturer. After the last wash, we carried out an extra washing step using H2O. We omitted the final elution step as we carried out the cDNA synthesis on the magnetic beads using a ScriptSeq v2 RNA-seq Library Preparation Kit (Illumina). The libraries were size selected using BluePippin DNA size selection system (Sage Science), and quality checked on Agilent High Sensitivity DNA Chips (Agilent Technologies). The pooled libraries were sequenced on Nextseq 500 (Illumina) DeepSeq at The University of Nottingham.
Bluepippin dna size selection system
Manufactured by Sage Science
Sourced in United States
The BluePippin DNA size selection system is a laboratory instrument designed to precisely separate DNA fragments based on their size. It utilizes pulsed-field gel electrophoresis technology to fractionate DNA samples with high resolution. The BluePippin system allows for automated, high-throughput DNA size selection, enabling researchers to isolate specific DNA fragments for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Apeki, by New England Biolabs (2 mentions)
Redtaq readymix, by Merck Group (2 mentions)
Miseq reagent kit v3, by Illumina (2 mentions)
T4 ligase, by New England Biolabs (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Epimark n6 methyladenosine enrichment kit, by New England Biolabs (1 mentions)
Rna fragmentation reagent, by Thermo Fisher Scientific (1 mentions)
Oligo dt magnetic beads, by New England Biolabs (1 mentions)
Murine rnase inhibitor, by New England Biolabs (1 mentions)
Agilent dna high sensitivity chip, by Agilent Technologies (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Scriptseq v2 rna seq library preparation kit, by Illumina (1 mentions)
Genomic dna kit, by Qiagen (1 mentions)
Qubit fluorimeter, by Thermo Fisher Scientific (1 mentions)
Nextera mate pair sample prep kit, by Illumina (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
P6 c4 chemistry, by Pacific Biosciences (1 mentions)
Miseq platform, by Illumina (1 mentions)
Truseq dna pcr free sample prep kit, by Illumina (1 mentions)
Genomic tip 20 g, by Qiagen (1 mentions)
10 protocols using bluepippin dna size selection system
1
m6A Methylation Profiling in Chicken Embryos
2
RADseq for Antarctic Usnea species
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RADseq libraries for Usnea antarctica U. aurantiacoatra RADseq library production, DNA isolations were pooled with sequence adapters (Rubin and Moreau 2016 (link)), subsequently digested with the restriction enzyme ApeKI (New England Biolabs, Ipswich, MA, USA) and ligated using T4 ligase (New England Biolabs). Up to 48 samples with compatible barcodes were pooled and selected for fragments of sizes between 300 and 500 bp using the BluePippin DNA size selection system (Sage Science, Beverly, MA, USA). The pooled libraries were amplified using the REDTaq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) prior to sequencing on an Illumina MiSeq using the MiSeq Reagent Kit v3 for 150 cycles (Illumina, San Diego, CA, USA) to produce single-end sequences with a length of 150 bp.
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3
Isolation and Genomic DNA Extraction
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C. cassiicola YN49 (CGMCC 3.20259) and CC01 (CGMCC 3.20258) strains were separately isolated from rubber plantations in Hekou, Yunnan province, and Yangjiang, Guangdong province. Both strains were maintained in our laboratory. Fungi were grown on potato dextrose agar medium and incubated at 28 °C for 10 days. Mycelia were harvested and DNA was extracted from grounded mycelia using a genomic DNA kit (Qiagen, New York, NY, USA). Agarose gel electrophoresis, a NanoDrop 1000 spectrophotometer (Thermo, Bedford, MA, USA) and a Qubit fluorimeter (Thermo, Bedford, MA, USA) were used to analyse the integrity, quality, and concentration of DNA, respectively. Genomic DNA was further purified for sequencing (Oxford Nanopore Technology, Oxford, UK) using a BluePippin DNA size selection system (Sage Science, Beverly, MA, USA).
4
Hybrid Genome Assembly Using Illumina and PacBio
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Sequencing was performed using the Illumina MiSeq platform and MiSeq Reagent Kit v2 (300 cycles), with MiSeq Reagent Kit v3 (600 cycles) being used for a more detailed analysis. Libraries for paired-end and mate-pair sequencing were prepared using a TruSeq DNA PCR-Free Sample Prep Kit and Nextera Mate Pair Sample Prep Kit (Illumina, San Diego, CA, USA), respectively. The generated reads were quality-filtered using Prinseq (36 (link)), and then assembled into contigs and scaffolds using SPAdes 3.9.0 (5 (link)). Gaps within and between scaffolds were closed by PCR amplification and sequenced using the ABI3730 Genetic Analyzer. In addition, sequence reads of 4–5 kb were generated on the PacBio RSII platform using a BluePippin DNA Size-Selection system (Sage Science, Beverly, MA, USA) and P6-C4 chemistry (Pacific Biosciences, Menlo Park, CA, USA). PacBio reads were used to verify the assembly and circularity of the scaffolds by mapping the reads using the BLASTn program of BLAST+ suite (8 (link)). MiSeq-read coverage was calculated by counting k-mers covering each scaffold in the assembly using SPAdes 3.9.0.
5
Genomic DNA Extraction and PacBio Sequencing
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Genomic DNA was extracted by a modified CTAB method as described in Maeda et al. [32 ]. Extracted DNA was purified with gravity-flow, anion-exchange tips (Genomic-Tip 20/G, Qiagen, Netherlands).
For PacBio sequencing, genomic DNA was amplified using a REPLI-g Single Cell Kit (Qiagen, Netherlands) after selection of long DNA (> 6 kbp) using the BluePippin DNA size-selection system (Sage Science, USA). To reduce artificial effects resulting from amplification, extracted gDNA was separated into six tubes before amplification, and each sample was amplified independently. After amplifying the entire genome, DNA was purified again with Genomic-Tips 20/G.
For PacBio sequencing, genomic DNA was amplified using a REPLI-g Single Cell Kit (Qiagen, Netherlands) after selection of long DNA (> 6 kbp) using the BluePippin DNA size-selection system (Sage Science, USA). To reduce artificial effects resulting from amplification, extracted gDNA was separated into six tubes before amplification, and each sample was amplified independently. After amplifying the entire genome, DNA was purified again with Genomic-Tips 20/G.
6
Whole-Genome Sequencing of Bacterial Isolates
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For the 25 isolates selected, whole-genome sequencing (WGS) was performed using Illumina MiSeq. Sequencing was performed at the University of Minnesota Mid-Central Research and Outreach Center (Willmar, MN) using a single 250-bp dual-index run on an Illumina MiSeq with Nextera XT libraries to generate approximately 30- to 50-fold coverage per genome. Several transconjugants were also sequenced using PacBio technology at the University of Minnesota Genomics Center (Minneapolis, MN). SMRTbell template libraries were generated from previously isolated unsheared raw genomic DNA using the Pacific Biosciences SMRTbell template preparation kit 1.0 (Pacific Biosciences, Menlo Park, CA). Finished DNA libraries subsequently were subjected to DNA size selection using the BluePippin DNA size selection system (Sage Science, Inc.) with a 7-kb cutoff to select DNA fragments larger than 7 kb. Sequencing was performed on the PacBio Sequel (Pacific Biosciences, Menlo Park, CA).
7
Serum Small RNA Sequencing Protocol
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RNA was extracted from serum using the Norgen Biotek Corp. plasma/serum RNA purification mini kit. Sequencing libraries were prepared from RNA using the Illumina TruSeq small RNA library preparation kit according to manufacturer’s instructions with the following modifications. Fifteen cycles were used for the final library PCR amplification step. The sequencing libraries were purified with the BluePippin DNA size selection system by Sage Science using the 3% agarose gel cassettes, gating from 125–150 bp. Prior to sequencing, library purification was confirmed by running samples on an Agilent Bioanalyzer using high-sensitivity DNA chips. Purified libraries were sequenced in multiplex using either the Illumina MiSeq or NextSeq platforms. Samples were multiplexed such that approximately 5 million single-ended 80-nt reads were sequenced for each sample.
8
RADseq Library Preparation Protocol
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RADseq libraries were prepared from the isolated DNA as described following Grewe et al. (2017 (link)). In summary, DNA isolations were pooled with sequence adapters (Rubin & Moreau, 2016 (link)), digested with the restriction enzyme ApeKI (New England Biolabs, Ipswich, MA, USA), and ligated using T4 ligase (New England Biolabs). All samples with compatible barcodes were pooled and selected for fragment sizes between 300 and 500 bp using the BluePippin DNA size selection system (Sage Science, Beverly, MA, USA). The pooled libraries were amplified using the REDTaq ReadyMix (Sigma‐Aldrich, St. Louis, MO, USA) prior to sequencing on an Illumina MiSeq using the MiSeq Reagent Kit v3 for 150 cycles (Illumina, San Diego, CA, USA) to produce single‐end sequences with a length of 150 bp.
9
Double-digest RAD-Seq Library Construction
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Following Poland et al. (2012) double-digest RAD-Seq libraries were constructed at the Institute for Integrative Systems Biology (IBIS; at Laval University) using the restriction enzyme pairing of SbfI and MspI. Uniquely barcoded samples were pooled into a library and then size selected for roughly 100-300 bp using a BluePippin® DNA size selection system (Sage Science Inc., Beverly, MA, USA). The library was forwarded to Génome Québec (Génome Québec Innovation Centre) and assessed using a LabChip GX (Perkin-Elmer, MA, USA) instrument to determine library size quality. The library was then sequenced as singleend, 100 base-pair reads on a single Illumina HiSeq2000 lane (Illumina, San Diego, CA, USA). All ddRADseq data will be archived on the Dryad Digital Repository upon acceptance.
10
PacBio Sequencing of Bacterial Genomes
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Nine strains were sequenced using PacBio technology at the Rochester Mayo Medical Genome Facility (Rochester, MN). SMRTbell template libraries were generated from previously isolated unsheared raw genomic DNA using the Pacific Biosciences SMRTbell template prep kit 1.0 (Pacific Biosciences, Menlo Park, CA). Finished DNA libraries were subsequently subjected to DNA size selection using the BluePippin DNA size selection system (Sage Science, Inc.), with a 7-kb cutoff to select DNA fragments greater than 7 kb. Sequencing was performed on the PacBio RSII (Pacific Biosciences) using P6 polymerase binding and C4 sequencing kits, with magnetic bead loading and 240-min acquisition.
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