High‐molecular‐weight DNA was extracted from a single pooled sample of insects (0.5 mL, ~50 individuals) using a previously established chloroform:isoamyl phase separation protocol (Jaworski et al., 2020 (link)). HMW DNA was size checked by Femto Pulse System (Agilent), and 10 μg of DNA was sheared to appropriate size range (15–20 kb) using Megaruptor 3 (Diagenode). The sheared DNA was concentrated by bead purification using PB Beads (PacBio). The sequencing library was constructed following the manufacturer's protocols using SMRTbell Express Template Prep Kit 2.0. The final library was size selected on a Pippin HT (Sage Science) using S1 marker with a 10–25 kb size selection. The recovered final library was quantified with Qubit HS kit (Invitrogen) and sized on Femto Pulse System (Agilent). The sequencing library was sequenced with PacBio Sequel II Sequencing kit 2.0, loaded to one 8 M SMRT cell, and sequenced in CCS mode for 30 h. RNA was extracted from similar pooled‐samples (N = 3) using the ZYMO (Irvine, CA, USA) Direct‐zol RNA miniprep kit (Cat. # R2050) and sequenced using NovaSeq (Illumina, San Diego, CA, USA) paired‐end (150 bp) sequencing performed by Novogene (Sacramento, CA, USA). RNA libraries were prepared by Novogene following their standard mRNA‐seq services (polyA capture followed by cDNA reverse transcription).
Femto pulse system
Manufactured by Agilent Technologies
Sourced in United States
The Femto Pulse System is a high-performance electrophoresis platform designed for the analysis of DNA, RNA, and protein samples. The system utilizes a proprietary femtosecond laser technology to provide rapid, high-resolution separation and detection of biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Phase lock gel, by Quantabio (7 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (6 mentions)
Smrtbell express template prep kit 2, by Pacific Biosciences (5 mentions)
Genomic dna 165 kb kit, by Agilent Technologies (5 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (5 mentions)
Quantus fluorometer quantifluor one dsdna dye assay, by Promega (5 mentions)
Pb beads, by Pacific Biosciences (4 mentions)
Megaruptor 3, by Diagenode (3 mentions)
Qbit 2.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Nanodrop spectrophotometer, by Agilent Technologies (3 mentions)
Megaruptor 2, by Diagenode (2 mentions)
Sageelf, by Sage Science (2 mentions)
Tissueruptor 2, by Qiagen (2 mentions)
G tube, by Covaris (2 mentions)
Quantifluor one dsdna dye assay, by Promega (2 mentions)
Smrtbell template prep kit, by Pacific Biosciences (2 mentions)
Pacbio sre kit, by Pacific Biosciences (2 mentions)
Nanobind tissue big dna kit, by Pacific Biosciences (2 mentions)
Monarch hmw dna extraction kit for cells blood, by New England Biolabs (2 mentions)
Qubit dna hs assay, by Thermo Fisher Scientific (2 mentions)
57 protocols using femto pulse system
1
Insect DNA and RNA extraction and sequencing
2
Extracting and Sequencing High-Molecular-Weight DNA
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High-molecular-weight DNA was provided to the University of Wisconsin–Madison Biotechnology Center DNA Sequencing Facility. The quality of the extracted DNA was measured on a Thermo Fisher Scientific NanoDrop One instrument. Concentrations, 260/230 ratios, and 260/280 ratios were logged. Quantification of the extracted DNA was measured using the Thermo Fisher Scientific Qubit dsDNA high-sensitivity kit. Samples were diluted before running on the Agilent FemtoPulse system to assess DNA sizing and quality. A PacBio HiFi library was prepared according to PN 101-853-100 version 03 (PacBio). Modifications include shearing with Covaris gTUBEs and size selection with Sage Sciences BluePippin. Library quality was assessed using the Agilent FemtoPulse system. The library was quantified using the Qubit dsDNA high-sensitivity kit. The library was sequenced on a PacBio Sequel II instrument using the sequel polymerase binding kit 2.2 at the University of Wisconsin—Madison Biotechnology Center DNA Sequencing Facility. Raw sequencing data were converted to circular consensus sequencing (CCS) FASTQ using SMRT Link version 8.0.
3
HiFi Library Preparation from Genomic DNA
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To generate a HiFi library, genomic DNA was sheared using the Megaruptor 2 (Diagenode) with a long hydropore and a 20-kb shearing protocol. Size distribution of the sheared DNA was characterized on the Femto Pulse system (Agilent Technologies) using the Genomic DNA 165 kb Kit. Sequencing libraries were constructed using the protocol “Preparing HiFi SMRTbell Libraries using SMRTbell Express Template Prep Kit 2.0” from PacBio. SMRTbells were size selected using 0.75% agarose 1–18 kb protocol on SageELF (Sage Science) according to the HiFi SMRTbell library protocol. Size-selected SMRTbells were examined on the Femto Pulse system (Agilent Technologies) using the Genomic DNA 165-kb Kit. Library fraction of 15 kb and 17 kb was selected for sequencing. Sequencing was performed on two SMRT cells using the Sequel II system and the 2.0 sequencing and binding chemistry, with 2 h pre-extension and 30 h movie time.
4
HiFi SMRTbell Library Preparation
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To generate a HiFi library, genomic DNA was sheared using the Megaruptor 2 (Diagenode) with a long hydropore and a 20 kb shearing protocol. Size distribution of the sheared DNA was characterized on the Femto Pulse system (Agilent Technologies) using the Genomic DNA 165 kb Kit. Sequencing libraries were constructed using the protocol "Preparing HiFi SMRTbell Libraries using SMRTbell Express Template Prep Kit 2.0" from PacBio. SMRTbells were size selected using 0.75% agarose 1-18kb protocol on SageELF (Sage Science) according to the HiFi SMRTbell library protocol. Size selected SMRTbells were examined on the Femto Pulse system (Agilent Technologies) using the Genomic DNA 165 kb Kit. Library fraction of 15 kb and 17 kb were selected for sequencing. Sequencing was performed on two SMRT cells using the Sequel II system and the 2.0 sequencing and binding chemistry, with 2 hours pre-extension and 30 hours movie time.
5
Isolation of High Molecular Weight Genomic DNA
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High molecular weight (HMW) genomic DNA (gDNA) was isolated from whole blood preserved in EDTA. 20 µL of whole blood was added to 2 ml of lysis buffer containing 10 mM Tris–HCl pH 8.0, 25 mM EDTA, 0.5% (w/v) SDS, and 100 µg/ml Proteinase K. Lysis was carried out at room temperature for a few hours until the solution was homogenous. The lysate was treated with 20 µg/ml RNase A at 37 °C for 30 min and cleaned with equal volumes of phenol/chloroform using phase lock gels (Quantabio, MA; Cat. # 2302830). DNA was precipitated by adding 0.4× volume of 5 M ammonium acetate and 3× volume of ice cold ethanol. The DNA pellet was washed twice with 70% ethanol and resuspended in an elution buffer (10 mM Tris, pH 8.0). Purity of gDNA was accessed using NanoDrop ND-1000 spectrophotometer and 260/280 ratio of 1.83 and 260/230 ratios of 2.34 was observed. DNA yield (120 µg total) was quantified using Qbit 2.0 Fluorometer (Thermo Fisher Scientific, MA). Integrity of the HMW gDNA was verified on a Femto pulse system (Agilent Technologies, CA).
6
Metagenome Sequencing Using PacBio HiFi Reads
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DNA was extracted from the bulk sediment sample (3.3 g) using a FastDNA spin kit for soil (MP Biomedicals; Irvine, CA, United States). The extracted DNA was purified and concentrated using NucleoBond HMW DNA (Takara Bio; Kusatsu, Shiga, Japan). The fragmentation level of the purified DNA was checked by pulsed-field capillary electrophoresis using Femto Pulse System (Agilent Technologies; Santa Clara, CA, United States). A SMRTbell library was prepared using the HMW DNA by SMRTbell Template Prep Kit v.2.0 (Pacific Biosciences; Menlo Park, CA, United States), and was size-selected on the BluePippin system using a 0.75% agarose cassette (Sage Science; Beverly, MA, United States) and a 5–30 kb high-pass cutoff. The size-selected SMRTbell library was bound to the sequencing polymerase enzyme using the Sequel II Binding Kit 2.1. Shotgun genomic DNA sequence data were collected on one run (with one SMRT Cell) of the PacBio Sequel II system with HiFi sequencing protocols and Sequencing Kit 2.0 chemistry (PacBio). HiFi reads (or CCS reads) were generated using ccs software v.10.01 with the default parameters (--minPasses 10 bp, --minPredictedAccuracy 0.0, and --maxLength 50,000 bp) and extracted >Q20.
7
High-Quality Genome Assembly from Nanopore and Hi-C Data
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Genomic DNA of AI was fragmented to a target size of 22 kb using a Megaruptor3 (Diagenode, Belgium). Fragmented DNA was then purified using AMPure PB (Pacific Biosciences, CA, USA). DNA fragment sizes were confirmed using a Femto Pulse System (Agilent Technologies). HiFi SMRTbell libraries were constructed using a SMRTbell Express Template Prep Kit 2.0 following the manufacturer’s protocol. Single-molecule sequencing was then conducted in circular consensus sequence (CCS) mode on a PacBio Sequel II platform. The quality of the obtained HiFi reads was confirmed based on k-mer (k = 40) spectra analysis with GeneScope.FK (https://github.com/thegenemyers/GENESCOPE.FK ) and PloidyPlot (https://github.com/thegenemyers/MERQURY.FK ), which are reimplemented and improved versions of GenomeScope 2.0 and SmudgePlot,27 (link) respectively. We confirmed that both HiFi reads and Omni-C reads were generated from a single diploid genome (Supplementary Fig. S1C and D ), with no contamination from other individuals or other species. HiFi reads were assembled using hifiasm version 0.13-r30828 (link) with default parameters. The primary unitigs (*.p_utg.gfa), which contain nucleotide sequences of a pair of both haplotypes, generated by hifiasm were used as contigs that were subsequently scaffolded with Omni-C data.
8
High Molecular Weight Genomic DNA Extraction
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High molecular weight (HMW) genomic DNA (gDNA) extraction and nucleic acid library preparation were carried out by the University of California Davis DNA Technologies Core (Davis, CA). A whole-body sample from a male and a female B. lynchi was homogenized in 500 µl of homogenization buffer (10 mM Tris–HCl, pH 8.0 and 25 mM EDTA) using TissueRuptor II (Qiagen, Hilden, Germany; Cat. # 9002755). 500 µl of lysis buffer (10 mM Tris, 25 mM EDTA, 200 mM NaCl, and 1% SDS) and proteinase K (100 µg/ml) were added to the homogenate and incubated overnight at room temperature followed by RNAse A (20 µg/ml) treatment at 37 °C for 30 min. The lysate was cleaned with equal volumes of phenol/chloroform using phase-lock gels (Quantabio, Beverley, MA; Cat. # 2302830) and the DNA was precipitated by adding 0.4× volume of 5 M ammonium acetate and 3× volume of ice-cold ethanol. The DNA pellet was washed twice with 70% ethanol and resuspended in an elution buffer (10 mM Tris, pH 8.0). The purity of the DNA was accessed using NanoDrop spectrophotometer (260/280 and 260/230 ratios) and the integrity of the HMW gDNA was verified on a Femto pulse system (Agilent Technologies, Santa Clara, CA).
9
Long-read Sequencing Protocol for MinION
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For long-read sequencing, DNA was quantified using a Qubit fluorometer (ThermoFisher) and quality checked on a Femto Pulse System with a genomic DNA 165-kb kit (Agilent, Santa Clara, CA, USA). A DNA aliquot of 8 µg was fragmented with a Covaris g-tube (Covaris Woburn, MA, USA) and small fragments were removed using Short Read Eliminator XS (Circulomics/PacBio, Menlo Park, CA, USA). The library for Oxford Nanopore MinIon sequencing was prepared using 1.69 µg of DNA and the LSK-109 ligation sequencing kit (Oxford Nanopore, Littlemore, UK), following repair and end-polishing of the sheared DNA using the NEBNext Companion Module for Oxford Nanopore Technologies ligation kit (New England Biolabs, Ipswich, MA, USA). Finally, 0.595 µg of library were loaded on a R9.4.1 MinION flow cell and sequencing was performed on a GridIon benchtop platform (Oxford Nanopore).
10
High-Molecular-Weight DNA Extraction from Plants
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High molecular weight (HMW) genomic DNA (gDNA) was extracted from 5 g of leaf tissue (Jcal-S-LA-6) using the Nanobind Plant Nuclei Big DNA Kit (Pacific Biosciences—PacBio, Menlo Park, California) and a plant-specific protocol (Workman et al. 2018 (link)) with the following modification. We used a nuclear isolation buffer supplemented with 350 mM Sorbitol to resuspend ground tissue and during the first wash of the nuclei pellet. The extracted HMW DNA was further purified using the high-salt-phenol-chloroform method (PacBio). The DNA purity (260/280 = 1.83 and 260/230 = 2.34) was assessed by absorbance ratios on a NanoDrop ND-1000 spectrophotometer. The DNA yield (47 ng/µL; 17 µg total) was quantified using QuantiFluor ONE dsDNA Dye assay (Promega, Madison, Wisconsin). The size distribution of the HMW DNA was estimated using the Femto Pulse system (Agilent, Santa Clara, California), and found that 51% of the total fragments were >90 kb.
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