Whole genomic DNA (gDNA) of Aci44 was extracted by modified Marmur procedure8 ,25 . Briefly, Aci44 was inoculated in CAMHB and incubated at 37 °C, 200 rpm, overnight. After incubation, Aci44 (3 mL) was harvested and lysed by 10 mg/mL lysozyme (Geneaid, Taiwan). RNA and protein were removed using 0.6 mg/mL RNase A (Serva, Germany) and 0.1 mg/mL proteinase K (Geneaid, Taiwan), respectively. DNA was purified using phenol–chloroform extraction. Quality and quantity of gDNA were determined by 1% agarose gel electrophoresis (Bio-rad, USA), UV spectrophotometer (OD260/280, OD260/230 ratio) (DeNovix DS-11 FX + spectrophotometer, DeNovix, USA), Qubit dsDNA BR assay kit (Invitrogen, USA), and Bioanalyzer using a high-sensitive DNA chip (Agilent, USA). One hundred ng of extracted DNA was used for library preparation using Ovation Ultralow System V2 (Nugen, Switzerland) and sequenced on NovaSeq (Illumina, USA) for short-read sequencing. Nanopore platform was applied for long-read sequencing. One µg of gDNA was used for library preparation by Ligation Sequencing kit (SQK-LSK110, Oxford Nanopore, UK) and sequenced on MinION Nanopore (Oxford Nanopore, UK).
High sensitive dna chip
Manufactured by Agilent Technologies
Sourced in United States
The High-sensitive DNA chip is a laboratory equipment designed for the detection and analysis of DNA samples. It utilizes advanced technology to provide high sensitivity in identifying and quantifying DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Bioanalyzer, by Agilent Technologies (2 mentions)
150 bp fragments, by Thermo Fisher Scientific (2 mentions)
Streptavidin magnetic beads, by Thermo Fisher Scientific (2 mentions)
Nextseq 500 system, by Illumina (2 mentions)
Agarose gel electrophoresis, by Bio-Rad (1 mentions)
Proteinase k, by Geneaid (1 mentions)
Rnase a, by Serva Electrophoresis (1 mentions)
Sqk lsk110 ligation sequencing kit, by Oxford Nanopore (1 mentions)
Ovation ultralow system v2, by Tecan (1 mentions)
Novaseq, by Illumina (1 mentions)
Ds 11 fx spectrophotometer, by DeNovix (1 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Lysozyme, by Geneaid (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Bioanalyser, by Agilent Technologies (1 mentions)
Quick rna miniprep kit, by Zymo Research (1 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (1 mentions)
5 protocols using high sensitive dna chip
1
Extraction and Sequencing of Aci44 Genomic DNA
2
Visualizing CRISPR-Cas9 Cutting by Gel Electrophoresis
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The size of the 18S rRNA gene fragments with and without CRISPR-Cas9 treatment was visualized by gel electrophoresis using a Bioanalyser (Agilent). Prior to loading into the gel, the Cas9-cut products (5 μL out of 10 μL) were treated with 1 mg/mL (final) Proteinase K (Invitrogen) at room temperature for 15 min to digest the Cas9 nuclease. Then, 1 to 2 μL of this proteinase-K-treated product was added into a well of an Agilent High Sensitive DNA Chip in a Bioanalyzer (Agilent) to visualize and verify cutting by CRISPR-Cas9.
3
Open Chromatin DNA Capture and Sequencing
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The isolated genomic DNAs (~200 ngs/25K cell) were sonicated into 150 bp fragments (Covaris) and the entire reaction product was mixed with 20 μL of Streptavidin magnetic beads (Invitrogen 65001, blocked using 0.1% cold fish gelatin in 1 × PBS overnight at 4°C) in 1 mL of B&W buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 M NaCl). Biotin-labeled open chromatin DNA was captured by streptavidin at 4°C for 2 hours with end-over-end rotation. The beads were washed four times with B&W buffer plus 0.05% of Triton X-100 followed by one-time wash with TE. The beads were resuspended in 50 μL of TE. The DNA was end-repaired, washed twice with B&W buffer plus 0.05% of Triton X-100, dA-tailed and washed with B&W buffer plus 0.05% of Triton X-100. And finally, NEB Illumina adaptor (NEB, E7370S) was ligated and washed twice with B&W buffer plus 0.05% of Triton X-100. A final wash of the bead bound DNA was performed with TE and the bound DNA was resuspended with 20 µls of TE. 10-20 μL of bound DNA was used for library amplification using PCR (NEB, E7370S). Routinely 8-10 PCR cycles were used to generate enough amount of library DNA for sequencing.
Low input cell numbers between 250-500, library was amplified 12-13 cycles. The library was examined and quantitated with high-sensitive DNA chip (Agilent, 5067-4627).
Low input cell numbers between 250-500, library was amplified 12-13 cycles. The library was examined and quantitated with high-sensitive DNA chip (Agilent, 5067-4627).
4
Biotin-labeled DNA Capture and Library Preparation
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The isolated Biotin-labeled genomic DNA from fixed reactions were sonicated into 150 bp fragments (Covaris) and 200 ng of DNA was mixed with 50 μl of Streptavidin magnetic beads (Invitrogen, 65001), previously blocked using 0.1% cold fish gelatin in 1 × PBS overnight at 4°C in 1 mL of B&W buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 M NaCl). Biotin-labeled open chromatin DNA was captured by streptavidin at 4°C for 2 hours with end-over-end rotation. The beads were washed four times with B&W buffer plus 0.05% of Triton X-100 followed by one wash with TE. The beads were resuspended in 50 μl of TE. The DNA was end-repaired, washed twice with B&W buffer plus 0.05% of Triton X-100, dA-tailed and washed with B&W buffer plus 0.05% of Triton X-100. And finally, NEB Illumina adaptor (NEB, E7370S) was ligated and washed twice with B&W buffer plus 0.05% of Triton X-100. A final wash of the bead bound DNA was performed with TE and the bound DNA was resuspended with 20 µls of TE. 10 μl of bound DNA was used for library amplification using PCR (NEB, E7370S). Routinely 8-10 PCR cycles were used to generate enough amount of library DNA for sequencing. The library was examined and quantitated with high-sensitive DNA chip (Agilent, 5067-4627). 4nM was loaded on NextSeq 500 System (Illumina).
5
RNA Sequencing of HUT 78 Cells
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RNA was extracted and purified from HUT 78 cells using Quick-RNA™ Miniprep Kit (Zymo Research, R1054). 1 µg of RNA was used to make libraries for RNA sequencing. Poly(A) mRNA was then isolated using NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, E7490S) and RNA sequencing was performed using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, E7760S) according to manufacturer's recommendations. The cDNA libraries were examined and quantitated with high-sensitive DNA chip (Agilent, 5067-4627). 4nM were loaded on NextSeq 500 System (Illumina).
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