After the indicated treatments, cells were harvested and subjected to the ChIP assay, according to the manufacturer’s instructions (EZ-magna ChIP A/G Chromatin Immunoprecipitation Kit or the HighCell# ChIP Kit). Chromatin fragmentation was performed using micrococcal nuclease (#88216; Thermo Fisher Scientific, Waltham, MA, USA) or chromatin shearing sonicator Bioruptor, while the binding elements for transcription factors were analyzed by quantitative real-time PCR using specific primer sets (see Table S8 ).
Bioruptor
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Bioruptor is a device used for efficient disruption and homogenization of biological samples, such as cells, tissues, or microorganisms. It utilizes high-intensity ultrasonic waves to achieve effective sample lysis and nucleic acid extraction.
Automatically generated - may contain errors
Lab products found in correlation
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Micrococcal nuclease, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Flow cell, by Illumina (1 mentions)
Hiseq 3000 4000 sbs kit, by Illumina (1 mentions)
Hiseq 4000 platform, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Synergy htx multi mode microplate reader, by Agilent Technologies (1 mentions)
Pierce coomassie plus bradford assay kit, by Thermo Fisher Scientific (1 mentions)
Neslab rte 7, by Thermo Fisher Scientific (1 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
Microfuge 22r, by Beckman Coulter (1 mentions)
6 protocols using bioruptor
1
ChIP Assay for Transcription Factor Analysis
2
DNA Fragmentation and Sequencing
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Isolated DNA was fragmented with Bioruptor (ThermoFisher Scientific, Waltham, MA, USA) to generate of approximately 300 bp library insert size. Quantity and quality control of the libraries were carried out with Qubit dsDNA HS Assay kit (ThermoFisher Scientific) and Agilent 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA, USA), respectively. High-quality DNA libraries were sequenced with the BGISEQ-500 platform (BGI-Tianjin) with read lengths of 100 bp.
3
Whole Genome Resequencing of Cultivated Genotypes
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The leaf material (0.2 g) was taken from two-week-old seedlings of all cultivated genotypes, snap-frozen with cryogenic liquid nitrogen, and preserved at an ultra-high-temperature of −80°C. A good quality gDNA was extracted using the cetyl trimethyl ammonium bromide (CTAB) protocol with slight modifications (Allen et al., 2006 (link)). The quantified gDNA was fragmented with Bioruptor (Thermo Fisher Scientific, USA) and 200–300 bp fragments were pooled-up. A sequencing library of each parent line gDNA was then prepared as follows: (1) the gDNA fragments were subjected to end-repairing and A-tailing; (2) the resulting fragments were ligated with bubble adapters that contained a sequential barcode, and then amplified with polymerase chain reaction (PCR). Next-generation sequencing (NGS, High-throughput Illumina SequencingTM 2500) based whole genome re-sequencing (>25× genome depth coverage) was performed at Beijing Genomics Institutes (BGI) company, Shenzhen, PR China.
4
DNA Library Construction and Sequencing
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The extracted DNA was fragmented with a Bioruptor (Thermo-Fisher Scientific, Waltham, MA, USA)) instrument to generate 200-300 bp fragments. The DNA libraries were constructed as follows: first, the DNA fragments were subjected to end repair and A tailing; second, the resulting DNA was ligated with bubble adapters that contained a barcode sequence and then amplified with PCR. Quality control was carried out with an Agilent 2100 (Agilent Technologies, Santa Clara, CA, USA) to assess the fragment size and a Qubit dsDNA HS Assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) to measure the DNA library concentrations. Qualified libraries were pooled together to form single-stranded DNA (ssDNA) circles, and then DNA nanoballs were generated with rolling circle replication. The final DNA nanoballs were loaded onto a sequencing chip and sequenced with a HiSeq platform (Illumina, Ca, USA).
5
Mouse DNA Methylation Profiling
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Three DNA samples were randomly selected from each treatment group (12 total DNA samples from individual mice processed separately) and sent on dry ice to Arraystar Inc. (Rockville, MD), with one sample per group as an input sample for sequence control. DNA was quantified by NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE), then fragmented to a range of 200–1,500 bp using a Diagenode Bioruptor, end-repaired, and 3' adenylated for ligation of genomic adapters. Fragments were immunoprecipitated by anti-5-methylcytosine antibody, then PCR amplified. AMPure XP beads were used to select fragments from 300–800 bp, then quantified by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Following denaturation with 0.1 M NaOH, single-stranded DNA molecules were captured and amplified in situ on an Illumina flow cell. Libraries were sequenced on the Illumina HiSeq 4000 platform using the HiSeq 3000/4000 SBS Kit (300 cycles) protocol, then Off-Line Basecaller (OLB V1.8) was used for base calling. Reads were aligned to the mouse genome (UCSC MM10) using HISAT2 (V2.1.0) (Kim et al., 2015 (link)) after passing the Solexa CHASTITY quality filter and resulting BED files were used for differential methylation analysis. Sequences have been made available at GEO accession number GSE137984.
6
Whole-Cell Protein Extraction from Thawed Cells
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Extraction of whole-cell protein from thawed cell pellets began by adding 100 μL of cold mammalian protein extraction reagent (M-PER) (Thermo Scientific, ThermoFisher Scientific, Cat#78501) per 10 μL of packed cell volume and 1 μL of 100× halt protease inhibitor cocktail (Thermo Scientific, ThermoFisher Scientific, Cat#87786) per 100 μL of M-PER. After resuspending cell pellets, lysates were sonicated on high for 5 min with 30 s on/off intervals at 4 • C (Diagenode Bioruptor; Thermo Scientific NESLAB RTE-7) to ensure proper lysis of nuclei. Samples were then rotated for 10 min at 4 • C, followed by centrifugation at 14 000g for 15 min at 4 • C (Beckman Coulter Microfuge 22R). Supernatants were transferred to separate tubes and stored at -80 • C for later use. Protein quantification was performed using the Pierce Coomassie Plus Bradford Assay Kit (Thermo Scientific, ThermoFisher Scientific, Cat#23236) with a working range of 125-1500 μg/mL and 96well microplates. Plates were read using a BioTek Synergy HTX Multimode Microplate Reader, which generated a standard curve and protein concentrations for all samples in Microsoft Excel.
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