PacBio HiFi data were generated from the HG00733 lymphoblastoid cell line as previously described (G. A. Logsdon et al., 2021 (link)) with modifications. Briefly, DNA was extracted from 4.3×10^6 cells using the Monarch HMW DNA Extraction Kit for Cells and Blood (New England Biolabs) with 1400 rpm lysis speed. After UV absorption and fluorometric quantification (Qubit High Sensitivity DNA kit, Thermo Fisher) on the DS-11 FX instrument (Denovix) and evaluation of DNA integrity on FEMTO Pulse (Agilent), 12 μg of DNA was prepared for sequencing using Megaruptor 3 shearing (Diagenode, settings 19/31) and the Express Template Prep Kit v2 and SMRTbell Cleanup Kit v2 (PacBio). The library was size-selected on a PippinHT instrument (Sage Science) using a 15 kbp high-pass cut. Five SMRT Cell 8Ms were run on a Sequel II instrument using Sequel II chemistry C2.0/P2.2 with 30-hour movie times, 2-hour pre-extension, and adaptive loading targets of 0.8–0.85 (PacBio). Circular consensus calling was performed with CCS version 6.0.0 (SMRT Link v.10.1) and reads with estimated quality scores ≥Q20 were selected for downstream analysis.
Monarch hmw dna extraction kit for cells and blood
Manufactured by New England Biolabs
The Monarch HMW DNA Extraction Kit for Cells and Blood is a laboratory equipment product designed to efficiently extract high molecular weight (HMW) DNA from various sample types, including cells and blood. The kit provides a simple and streamlined workflow to isolate intact, high-quality DNA suitable for downstream applications such as long-read sequencing or other genomic analyses.
Automatically generated - may contain errors
Lab products found in correlation
Ds 11 fx instrument, by DeNovix (2 mentions)
Megaruptor 3 shearing, by Diagenode (2 mentions)
Express template prep kit v2 and smrtbell cleanup kit v2, by Pacific Biosciences (2 mentions)
Sequel 2 chemistry c2 0 p2, by Pacific Biosciences (2 mentions)
Qubit high sensitivity dna kit, by Thermo Fisher Scientific (2 mentions)
Pippinht instrument, by Sage Science (2 mentions)
Femto pulse, by Agilent Technologies (2 mentions)
Tapestation, by Agilent Technologies (1 mentions)
3 protocols using monarch hmw dna extraction kit for cells and blood
1
PacBio HiFi Sequencing of HG00733
2
Extraction of High Molecular Weight DNA
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High-molecular weight (HMW) DNA was extracted using the NEB Monarch HMW DNA Extraction kit for Cells and Blood (Catalog #T3050L) according to the kit manufacturer’s instructions. Briefly, for cells maintained in tissue culture, cells were trypsinized until detached and then centrifuged at 1000 g for three minutes before adding prep and lysis solution according to the manufacturer’s instructions. Cells were incubated for 10 minutes at 1800 RPM in a thermomixer and DNA from the lysed cells was precipitated onto glass beads, washed twice with 80% ethanol, and finally eluted in Monarch Elution Buffer II according to manufacturer’s instructions. DNA was quantified using a Qubit 4 fluorometer and the Qubit BR dsDNA quantification reagents (Catalog #Q32850). For DNA extraction from peripheral blood, red blood cell lysis was first performed prior to DNA extraction according to the manufacturer’s instructions. DNA quality was assessed by Nanodrop and the average molecular weight was verified to be 60 kb or larger using an Agilent Tapestation (Catalog #5067–5365, 5067–5365).
3
HiFi Sequencing of HG00733 Cell Line
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PacBio HiFi data were generated from the HG00733 lymphoblastoid cell line as previously described (Logsdon et al. 2021 (link)) with modifications. Briefly, DNA was extracted from 4.3 × 106 cells using the Monarch HMW DNA Extraction Kit for Cells and Blood (New England Biolabs) with 1400 rpm lysis speed. After UV absorption and fluorometric quantification (Qubit High Sensitivity DNA kit, Thermo Fisher Scientific) on the DS-11 FX instrument (Denovix) and evaluation of DNA integrity on FEMTO Pulse (Agilent), 12 μg of DNA was prepared for sequencing using Megaruptor 3 shearing (Diagenode, settings 19/31) and the Express Template Prep Kit v2 and SMRTbell Cleanup Kit v2 (PacBio). The library was size-selected on a PippinHT instrument (Sage Science) using a 15 kbp high-pass cut. Five SMRT Cell 8Ms were run on a Sequel II instrument using Sequel II chemistry C2.0/P2.2 with 30-h movie times, 2-h pre-extension, and adaptive loading targets of 0.8–0.85 (PacBio). Circular consensus calling was performed with CCS version 6.0.0 (SMRT Link v.10.1) and reads with estimated quality scores ≥Q20 were selected for downstream analysis.
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