For whole-exome sequencing, 150 ng of sample DNA was sheared to 300 bp length using Covaris microTUBEs (Covaris, Woburn, MA, USA). Library construction was carried out with the KAPA HyperPrep Kit (Roche, Basel, Switzerland) with IDT xGEN® Dual Index UMI Adapters (IDT, Coralville, IA, USA) and four PCR cycles. Afterwards, exon capturing using the IDT xGEN® Exom Research Panel v1.0 (IDT, Coralville, IA, USA) was carried out according to manufacturer’s protocol. The library was size-selected to 350–560 bp using the BluePippin device (Sage Science, Beverly, MA, USA), quantified using Qubit dsDNA HS (ThermoFisher, Waltham, MA, USA), evaluated via capillary electrophoresis using Bioanalyzer (Agilent, Santa Clara, CA, USA), and ultimately sequenced on Illumina NextSeq 500 sequencer with 2 × 150 bp read length.
Bluepippin device
Manufactured by Sage Science
Sourced in United States
The BluePippin is a lab device used for automated size-selection of DNA samples. It utilizes pulsed-field gel electrophoresis to separate DNA fragments by size and can be programmed to collect specific size ranges. The BluePippin is designed to provide precise and consistent DNA size selection for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Qubit dsdna hs, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 sequencer, by Illumina (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Microtube, by Covaris (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Nextera dna library preparation kit, by Illumina (1 mentions)
Megaruptor, by Diagenode (1 mentions)
Qubit 4.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Smrtbell template prep kit, by Pacific Biosciences (1 mentions)
Pacbio sequel sequencer, by Pacific Biosciences (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Femto pulse, by Agilent Technologies (1 mentions)
G tube, by Covaris (1 mentions)
Smrtbell express template prep kit, by Pacific Biosciences (1 mentions)
Megaruptor 2, by Diagenode (1 mentions)
Blunt ta ligase master mix, by New England Biolabs (1 mentions)
Ultra 2 end prep module, by New England Biolabs (1 mentions)
Sqk lsk108 kit, by Oxford Nanopore (1 mentions)
Sqk lsk108, by Oxford Nanopore (1 mentions)
Qubit fluorometer dsdna broad range assay, by Thermo Fisher Scientific (1 mentions)
6 protocols using bluepippin device
1
Whole-Exome Sequencing Protocol with Targeted Enrichment
2
PacBio and Illumina Sequencing of Genomic DNA
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For PacBio SMRT sequencing, the gDNA was sheared using a Megaruptor device (large hydropore, Megaruptor, Diagenode, shearing size 35 kb), used for PacBio SMRT library preparation (SMRTbell Temp Prep Kit 1.0, Pacific Biosciences). Size selection was done on a BluePippin device (0.75% DF marker S1 high-pass 15-20 kb, Sage Science). The prepared samples were sequenced on a PacBio RSII instrument (DNA/Polymerase Binding Kit P6 v2, DNA Sequencing Kit 4.0 v2, Pacific Biosciences), the mean read length was 13.7 kb. One SMRT-cell was used for the KO sample (229x coverage) and 2 SMRT-cells were run for the WT sample (140x and 95x coverage). For Illumina sequencing, libraries were prepared with the Nextera DNA Library Preparation kit and sequenced on an Illumina MiSeq instrument (MiSeq Reagent Kit v2 Nano, PE250 (paired end 250 bp), 500 Mb), with an average of 55-56x coverage per genome.
3
PacBio Whole Genome Sequencing and Assembly
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A genomic DNA library was constructed using a SMRTbell Template Prep kit (Pacific Biosciences, CA, USA) in accordance with the manufacturer’s protocol. A BluePippin device (Sage Science, Inc., Beverly, MA, USA) was used to select 20 kb insert size fragments for the SMRTbell Template library. Quality inspection and quantification of the size-selected library were done using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit 4.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Prepared whole-genome libraries were sequenced on a PacBio Sequel sequencer (Pacific Biosciences, Menlo Park, CA, USA) with one SMRT cell at the Engineering Research Center of the Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Changchun, China. The genome was assembled using SMARTdenovo as described below, in accordance with www.github.com/smartdenovo . The completeness of the assembled genome was evaluated using the Core Eukaryotic Genes Mapping Approach (CEGMA) [19 (link)] and Benchmarking Universal Single-Copy Orthologs (BUSCO v3) [20 (link),21 (link)] with conserved orthologous gene profiles for fungi.
4
SMRT Bell Library Preparation
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A SMRT bell library was produced using the SMRTbell Express Template Prep Kit (Pacific Biosciences 101-357-000). 10 μg of gDNA were mechanically sheared to an average size distribution of 15-20 kb, using a Covaris gTube (Covaris p/n 520079). 3 μg of sheared gDNA was DNA damage repaired and end-repaired using polishing enzymes. A ligation reaction was performed to create the SMRT bell template, according to the manufacturer’s instructions. A Blue Pippin device (Sage Science) was used to size select the SMRT bell template and enrich the big fragments beyond 10 kb. The sized selected library was quality inspected and quantified using a Femto Pulse (Agilent) gDNA analysis assay and on a Qubit Fluorometer respectively. A ready to sequence SMRT bell-Polymerase Complex was created using the P6 DNA/Polymerase binding kit 2.0 (Pacific Biosciences p/n 100-236-500) according to the manufacturer instructions. The Pacific Biosciences RSII instrument was programmed to load and sequence the sample on 1 SMRT cell v3.0 (Pacific Biosciences p/n100-171-800), taking 1 movie of 360 minutes. A MagBead loading (PacBio p/n 100-133-600) method was chosen in order to improve the enrichment of the longer fragments.
5
Nanopore Sequencing of Genomic DNA
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Genomic DNA (5 µg) was sheared to ~5–25-kilobase fragments using Megaruptor® 2 (Diagenode, B06010002) and was then size-selected (10–30 kilobases) with a BluePippin device (Sage Science, MA) to remove the small DNA fragments. Subsequently, genomic libraries were prepared using the Ligation Sequencing 1D Kit (SQK-LSK108, Oxford Nanopore, UK). End-repair and dA-tailing of DNA fragments were performed using the Ultra II End Prep module (New England Biolabs, E7546L), according to manufacturer’s recommended protocols. Finally, the purified dA-tailed sample was incubated with blunt/TA ligase master mix (#M0367, NEB), tethered with 1D adapter mix from the SQK-LSK108 Kit (Oxford Nanopore Technologies), and purified. The resulting library was sequenced on R9.4 flow cells using GridION X5.
6
SMRT Bell Template Preparation
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SMRT bell templates were produced using the DNA Template Prep Kit 1.0 (Pacific Biosciences, Menlo Park, USA) (p/n 100-259-100). The input genomic DNA concentration was measured using a Qubit Fluorometer dsDNA Broad Range assay (Thermo Fischer) (p/n 32850), revealing a concentration of 88 ng/μl (total amount 25.2 μg). A total of 10 μg of gDNA was mechanically sheared to an average size distribution of 15 kbp, using a Megaruptor Device (Diagenod, Seraing, Belgium) . A Bioanalyzer 2100 12K DNA Chip assay (Agilent, Santa Clara, USA) (p/n 5067-1508) was used to assess the fragment size distribution. Five μg of sheared gDNA was DNA damage repaired and end-repaired using polishing enzymes. A bluntend ligation reaction followed by exonuclease treatment was performed to create the SMRT bell template. A Blue Pippin device (Sage Science, Beverly, USA) was used to size-select the SMRT bell template and enrich large fragments >7 kbp. The sized-selected library was quality (p/n 100-133-600) method was chosen in order to improve the enrichment the longer fragments.
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