Re-sequencing was performed to detect the off target in edited strains. Total DNA was extracted from C. glutamicum according to manufacturer’s protocol (TIANGEN, Beijing, China). DNA quality was determined using Qubit Fluorometer (Thermo Fisher Scientific, San Jose, CA, USA) to determine total mass and Fragment Analyzer to determine DNA integrity. The genome of C. glutamicum was sequenced using an Illumina HiSeq 4000 system (Illumina, San Diego, CA, USA) at the Beijing Genomics Institute (Shenzhen, China). Genomic DNA was sheared randomly to construct three read libraries with lengths of (300 bp) by a Bioruptor ultrasonicator (Diagenode, Denville, NJ, USA) and physico-chemical methods. The paired-end fragment libraries were sequenced according to the Illumina HiSeq 4000 system’s protocol. Raw reads of low quality from paired-end sequencing (those with consecutive bases covered by fewer than five reads) were discarded. The sequenced reads were assembled using SOAPdenovo v1.05 software.
Bioruptor ultrasonicator
Manufactured by Diagenode
Sourced in United States, Belgium
The Bioruptor ultrasonicator is a laboratory instrument used for the fragmentation of biological samples, such as DNA, chromatin, and proteins, through the application of high-frequency sound waves. The device generates controlled sonication conditions to achieve reproducible and efficient sample disruption.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 4000 system, by Illumina (7 mentions)
Hiseq 4000, by Illumina (6 mentions)
Dynabeads mrna direct purification kit, by Thermo Fisher Scientific (4 mentions)
Epimark n6 methyladenosine enrichment kit, by New England Biolabs (4 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Pico input strand specific total rna seq, by Illumina (2 mentions)
Dna lobind tube, by Eppendorf (1 mentions)
Ab4729, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
Ab8895, by Abcam (1 mentions)
Ipure kit v2, by Diagenode (1 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Nebnext ultra dna library prep kit, by Illumina (1 mentions)
Rsii platform, by Pacific Biosciences (1 mentions)
Gaiix platform, by Illumina (1 mentions)
Quick blunting kit, by New England Biolabs (1 mentions)
Dna template prep kit 3, by Pacific Biosciences (1 mentions)
Epiquik tissue chip kit, by Epigentek (1 mentions)
Hiseq 3000 sequencer, by Illumina (1 mentions)
26 protocols using bioruptor ultrasonicator
1
Genome Resequencing of C. glutamicum
2
Profiling the m⁶A Methylome of T2D Islets
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To profile the m6A methylome of the T2D islets, we applied m6A-seq (m6A-MeRIP-seq) to 7 T2D patients and 8 non-diabetic controls as well as one prediabetic sample and one sample from an adolescent non-diabetic. A minimum of 20,000 human islets equivalents (IEQs)/patient were used for total RNA extraction using Trizol according to the manufacturer’s instruction. mRNA was isolated from total RNA using Dynabeads mRNA DIRECT purification kit (Thermo Fisher, USA). mRNA was adjusted to 15ng/ul in 100ul and fragmented using Bioruptor ultrasonicator (Diagenode) with 30s on/off for 30 cycles. m6A-immunoprecipitation (m6A-IP) and library preparation were performed using EpiMark N6-Methyladenosine enrichment kit (NEB, USA). Input and RNA eluted from m6A-IP were used to prepare libraries with KAPA mRNA Hyper Kit (patient islets samples).
In the samples from EndoC-βH1 cells and mouse islets, we sequenced three replicates for each experimental group. Total RNA was purified using Trizol according to the manufacturer’s instruction. Four μg of sonicated total RNA were used for m6A-IP and library were constructed using Takara Pico-Input Strand-Specific Total RNA-seq for Illumina.
All sequencing was performed on Illumina HiSeq 4000 according to the manufacturer’s instructions. Approximately 30 million single-end 50-bp reads were generated for each sample.
In the samples from EndoC-βH1 cells and mouse islets, we sequenced three replicates for each experimental group. Total RNA was purified using Trizol according to the manufacturer’s instruction. Four μg of sonicated total RNA were used for m6A-IP and library were constructed using Takara Pico-Input Strand-Specific Total RNA-seq for Illumina.
All sequencing was performed on Illumina HiSeq 4000 according to the manufacturer’s instructions. Approximately 30 million single-end 50-bp reads were generated for each sample.
3
Profiling the m⁶A Methylome of T2D Islets
4
Illumina Genome Sequencing and Assembly
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Genome sequencing was performed on an Illumina HiSeq 4,000 system (Illumina) at BGI company (Shenzhen, China). Genomic DNA was sheared randomly to construct three read libraries with lengths of 300 bp by a Bioruptor ultrasonicator (Diagenode, Denville, NJ, United States) and physico-chemical methods. The paired-end fragment libraries were sequenced according to the Illumina HiSeq 4,000 system’s protocol. Raw reads of low quality from paired-end sequencing (those with consecutive bases covered by fewer than five reads) were discarded. The sequenced reads were assembled using SOAPdenovo v1.05 software. The completeness and contamination of the genomes were accurately estimated by the CheckM pipeline.
5
ChIP-qPCR Analysis of Histone Modifications
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The cells used for the ChIP assay were cultured to about 80% confluence, and then, 1% formaldehyde and 0.125 M glycine were added to the media to initiate and terminate crosslinking, respectively. The scraped cells were placed in a 1.5‐ml DNA LoBind Tube (Eppendorf) and centrifuged at 2500 g and 4°C for 5 min. The pellet was then resuspended with nuclei lysis buffer (50 mM Tris‐Cl, 10 mM EDTA, 1% SDS, and a protease inhibitor cocktail) and the released chromatin was sonicated using a Bioruptor ultrasonicator (Diagenode) before being diluted with IP dilution buffer (20 mM Tris‐Cl, 2 mM EDTA, 150 mM NaCl, 1% Triton X‐100 and a protease inhibitor cocktail). The samples were incubated with specific antibodies overnight at 4°C. The antibodies were anti‐H3K4me1 (ab8895, Abcam), anti‐H3K27ac (ab4729, Abcam), and anti‐H3 (ab1791, Abcam). And the chromatin–antibody complexes were captured by protein A/G beads. We used qPCR to assay the immunoprecipitated DNA. The primers used in this analysis are in Table S1 .
6
Bacterial Genome Sequencing and Assembly
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Bacterial genomic DNA was sheared randomly to construct three read libraries with lengths of 250 bp by a Bioruptor ultrasonicator (Diagenode, Denville, NJ, USA) and physicochemical methods. The paired-end fragment libraries with an insert size of 270 bp were sequenced according to the Illumina HiSeq 4000 system protocol at the Beijing Genomics Institute (Shenzhen, China). Low quality raw reads (>40% of the bases with Q value ≤20 or containing >10% ambiguous bases) were discarded. The remaining high-quality reads were assembled to form scaffolds using SOAP denovo v1.05 software. A summary of the genome assembly statistics for each genome is provided in Table S1.
7
STAT1 ChIP-seq in Pulmonary F4/80+ Cells
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Pulmonary F4/80+ cells (5 x 106) isolated from immunized mice were crosslinked, lysed, and sonicated with the Bioruptor ultrasonicator (Diagenode, Denville, NJ). 10% of chromatin was reserved as input control. The remaining chromatin was immunoprecipitated with antibodies against Normal IgG or STAT1 (Cell Signaling Technology, Danvers, MA) bound to Protein A/G Magnetic Beads (Pierce Biotechnology, Rockford, IL). Immunoprecipitated DNA and Input DNA was reverse crosslinked, eluted, and purified with the IPure kit v2 (Diagenode) according to manufacturer’s instructions.
8
Quantifying Cellular Actin Fractions
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80–85% confluent cells were washed directly on 15 cm petri dishes twice with PBS at room-temperature, scraped into PBS and pelleted. Cell pellets were resuspended in 800 µl of 37°C lysis buffer (1 mM ATP, 50 mM PIPES pH 6.9, 50 mM NaCl, 5 mM MgCl2, 5 mM EGTA, 5% vol/vol glycerol and 0.1% vol/vol for: Nonidet P-40, Tween 20, and Triton X-100) supplemented with protease inhibitors. Cells were homogenized by gently pipetting 10 times up and down and incubated at 37°C for 30 min. Samples were centrifuged at 16000 g for 75 min at 22–25°C. Supernatants were removed for determination of G-actin. Pellets containing F-actin were mixed with 800 µl of RIPA buffer and sonicated on ice for 5 min (30 sec. ON/30 sec OFF, max power) using Bioruptor ultrasonicator (Diagenode, Belgium) to dissolve them. Twenty microliters of each fraction were mixed with SDS-loading buffer, denatured for 8 minutes at 95°C and loaded on polyacrylamide gel. Actin detection was performed using an anti-actin monoclonal antibody (code sc-8432) purchased from Santa Cruz, Germany.
9
Genome Sequencing of Mutant Strain N-11
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The kit method (Sangon, China) was used to extract the genomic DNA of the mutant strain N-11. The genome of N-11 was sequenced using an Illumina HiSeq 4000 system (Illumina, San Diego, CA, USA) at the Beijing Genomics Institute (Shenzhen, China). Genomic DNA was sheared randomly to construct three read libraries with lengths of 3,979,503 by a Bioruptor ultrasonicator (Diagenode, Denville, NJ, USA) and physico-chemical methods. The paired-end fragment libraries were sequenced according to the Illumina HiSeq 4000 system's protocol. Raw reads of low quality from paired-end sequencing (those with consecutive bases covered by fewer than ve reads) were discarded. The sequenced reads were assembled using SOAP denovo v1.05 software.
10
Marfan Syndrome Genetic Screening Protocol
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