High molecular mass genomic DNA was purified using the MagAttract HMW DNA kit (Qiagen). For CLR library preparation, at least 5 μg of high molecular mass genomic DNA (more than 50% of fragments ≥40 kb) was sheared to approximately 40 kb using the Megaruptor 3 (Diagenode B06010003), followed by DNA repair and ligation of PacBio adapters using the PacBio SMRTbell Express Template Prep Kit 2.0 (100–938-900). Libraries were then size-selected for >30 kb using a BluePippin instrument with 0.75% agarose cassettes (Sage Science). After quantification with the Qubit dsDNA High Sensitivity assay (Thermo Q32854), libraries were diluted to 50 pM per SMRT cell, hybridized with PacBio V2 sequencing primer, and bound with SMRT seq polymerase using Sequel II Binding Kit 2.0 (PacBio 101–842-900). CLR sequencing was performed on the Sequel II instrument using 8M SMRT Cells (101–389-001) and Sequel II Sequencing 2.0 Kit (101–820-200), with a 15 h movie time per SMRT cell. Initial quality filtering, basecalling, and adaptor marking were done automatically on board the Sequel II.
Agarose cassettes
Manufactured by Sage Science
The 0.75% agarose cassettes are a type of lab equipment designed for gel electrophoresis applications. They are pre-cast gel trays that contain 0.75% agarose, a common matrix used for separating and analyzing DNA, RNA, and protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Megaruptor 3, by Diagenode (2 mentions)
Qubit dsdna high sensitivity assay, by Thermo Fisher Scientific (2 mentions)
Smrtbell express template prep kit 2, by Pacific Biosciences (2 mentions)
Sequel 2 binding kit 2, by Pacific Biosciences (2 mentions)
Magattract hmw dna kit, by Qiagen (1 mentions)
Bluepippin instrument, by Sage Science (1 mentions)
Pippinht, by Sage Science (1 mentions)
Sequel 2 sequencing 2.0 kit, by Pacific Biosciences (1 mentions)
2 protocols using agarose cassettes
1
Workflow for Preparing High Molecular Weight DNA Library
2
PacBio CCS Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For CCS library preparation, ≥3 ug of high molecular weight genomic DNA (more than 50% of fragments ≥40 kb) was sheared to ~15 kb using the Megaruptor 3 (Diagenode B06010003), followed by DNA repair and ligation of PacBio adapters using the PacBio SMRTbell Express Template Prep Kit 2.0 (100-938-900) and removal of incomplete ligation products with the SMRTbell Enzyme Clean Up Kit 2.0 (PacBio 101-938-500). Libraries were then size-selected for 15 kb +/− 20% using the PippinHT with 0.75% agarose cassettes (Sage Science). Following quantification with the Qubit dsDNA High Sensitivity assay (Thermo Q32854), libraries were diluted to 60 pM per SMRT cell, hybridized with PacBio V5 sequencing primer, and bound with SMRT seq polymerase using Sequel II Binding Kit 2.2 (PacBio 101-908-100). CCS sequencing was performed on the Sequel iIe instrument using 8M SMRT Cells (101-389-001) and Sequel II Sequencing 2.0 Kit (101-820-200), utilizing PacBio’s adaptive loading feature with a 2 hour pre-extension time and 30 hour movie time per SMRT cell. Initial quality filtering, basecalling, adapter marking, and CCS error correction was done automatically on board the Sequel iIe.
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