6 μg of genomic DNA was diluted in 30 μl milliQ and subsequently fragmented by sonication in icecold water in the Biorupter (Diagenode, Liège, Belgium) for 1 to 4 cycles of 15 times 10 sec ON–10 sec OFF (and briefly spinned down after each cycle) until the fragment size was between 100–600 bp, with the majority of the fragments around 250 bp. Fragment size was verified by electrophoresis on a 2% agarose gel (A9539, Sigma-Aldrich, St. Louis, MO, USA).
Biorupter
Manufactured by Diagenode
Sourced in Belgium
The Biorupter is a laboratory equipment designed for the disruption and homogenization of biological samples. It utilizes high-frequency sound waves to break down cellular structures and release cellular contents. The Biorupter is a versatile tool that can be used in a variety of applications, including DNA, RNA, and protein extraction, as well as cell lysis and sample preparation for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protein g dynabead, by Thermo Fisher Scientific (5 mentions)
Benzonase, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Novaseq, by Illumina (3 mentions)
Pr 619, by LifeSensors (2 mentions)
1 10 phenanthroline, by LifeSensors (2 mentions)
Otub1, by LifeSensors (2 mentions)
Pierce anti ha magnetic beads, by Thermo Fisher Scientific (2 mentions)
Nebnext ultra 2 dna library prep kit, by Illumina (2 mentions)
Antibody coupling kit cat 14311d, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Fast library prep kit, by Illumina (2 mentions)
Pierce magnetic chip kit, by Thermo Fisher Scientific (2 mentions)
A9539, by Merck Group (1 mentions)
Microplex kit, by Diagenode (1 mentions)
Nextseq 500, by Illumina (1 mentions)
α h3k27ac, by Abcam (1 mentions)
Anti myc antibody, by Merck Group (1 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
47 protocols using biorupter
1
DNA Fragmentation by Sonication
2
ChIP-seq of Histone Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP of H3K27ac, H3K4me3, and H3K4me1 was done as described in [6 (link)]. In brief, cells were fixed with 1 % formaldehyde; lysed in 50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS, protease inhibitors, and Na-butyrate; and sonicated in a Biorupter (Diagenode) into ~200 base-pair fragments. After sedimentation, the supernatant was diluted 10 times and chromatin incubated with anti-H3K27ac (Diagenode c15410174), anti-H3K4me3 (Diagenode c15410003) or anti-H3K4me1 (Diagenode c15410037) antibodies, each at 2.5 μg/106 cells, for 2 h at 4°C. ChIP samples were washed, cross-links reversed, and DNA eluted for 2 h at 68°C. DNA was purified using phenol-chloroform isoamylalcohol and dissolved in H2O. Libraries were prepared using a Microplex kit (Diagenode) and sequenced on a Nextseq 500 or Novaseq (Illumina).
3
Immunoprecipitation and DUB Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 1×107 HEK293 cells were washed, pelleted and stored at −80°C for each immunoprecipitation. The pellets were resuspended in 1 ml IP buffer B (20 mM HEPES-KOH pH 7.4, 110 mM potassium acetate, 2 mM MgCl2, 0.1% v/v Tween-20, 0.1% v/v Triton X-100, 150 mM NaCl, 1 mM DTT, 0.1 mM PTSF) containing 1x cOmplete Protease Inhibitor Cocktail, 20 μM PR-619 (LifeSensors, Cat#: SI9619–5X5MG), 5 mM 1,10-phenanthroline (LifeSensors, Cat#: SI9649), and 1 μl/ml Benzonase (Sigma-Alrich). Samples were incubated on ice for 30 min, sonicated with a Diagenode Biorupter on low setting for 30 s on and 30 s off for five rounds at 4°C and spun at max speed (21,130 g) for 5 min at 4°C. 925 μl of sample was added to 100 μl washed Pierce Anti-HA Magnetic beads (Thermo Fisher). Sample was incubated with beads rotating for 2 h at 4°C, washed 3x in IP buffer B, resuspended in 100 μl of IP buffer B and split into three 30 μl aliquots. 1 μl of 20 mM PR-619 was added to sample 1 (untreated), 1 μl of USP2 (LifeSensors, Cat#: DB501) was added to sample 2 (DUBPAN) and 2 μl of OTUB1 (LifeSensors, Cat#: DB201) was added to sample 3 (DUBK48). Samples were incubated at 30°C for a minimum of 1 h. Samples were eluted by addition of 10 μl 4x LDS sample buffer with 100 mM DTT and boiling at 95°C for 10 min for further processing by immunoblotting.
4
Profiling H3K27ac ChIP-seq in Kasumi-1 AML1-ETO Cells
5
Transcriptional Regulation Analysis by ChIP-seq
6
ChIP Protocol for C. albicans Transcriptome Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Chromatin immunoprecipitation (ChIP) protocol was adapted from Nobile et al. (2012) [54 (link)]. C. albicans cells were grown in liquid YPD to stationary phase at 30°C, and then collected and washed with PBS twice. 5 x 106 cells were inoculated into 200 mL of YPD and cultured to OD600 = 0.4. Cells (10 mL) were transferred to a 9 mm-dish and treated in 5% CO2 at 37°C with shaking for 6 hours, and then fixed and cross-linked with 1% formaldehyde at room temperature. The cross-linking reaction was quenched after 20 min by adding glycine to a final concentration of 125 mM. The cells were harvested, resuspended in ice-cold lysis buffer, and homogenized with a bead beater. Sonication was performed with a Diagenode Biorupter (15 min, high setting, 30 sec on, 1 min off) to obtain chromatin fragments of an average size of 500–1000 bp. The chromatin was immunoprecipitated with 2 μg of anti-Myc antibody (Millipore, Inc.) and protein A-Sepharose beads (GE Healthcare). ChIP DNA was analyzed by quantitative real-time PCR assays.
7
Sequential Chromatin Immunoprecipitation Assay
8
Chromatin Isolation Protocol for Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After sorting, cells were adjusted to 2×106 cells/ml in 3%–5% FCS in PBS. Cells were fixed with 1% formaldehyde for 12 minutes at room temperature. Fixation was stopped by adding glycine to a final concentration of 0.25M. Cells were collected by centrifugation at 1000 g for 5 minutes, washed twice with cold PBS, and frozen as a pellet on dry ice and stored at −80°C. Chromatin was prepared as previously described (Dorsett et al., 2014 (link); Swain et al., 2016 (link)) with slight modifications. To isolate chromatin, 10×106 cells were suspended in 300 mL sonication buffer [0.4% SDS, 10mM Tris pH 7.6] with protease inhibitors (Roche cOmplete, EDTA-free Protease Inhibitor Cocktail) and incubated on ice for 20 minutes. The cell suspension was sonicated, and chromatin was sheared to a range of 200–1000bp using a Diagenode Biorupter. After sonication, 900 μL of adjustment buffer [10mM Tris pH 7.6, 1.33 mM EDTA pH 8, 0.133% Na deoxycholate, 1.33% Triton] plus protease inhibitors was added and chromatin was incubated on ice for an additional 20 minutes. Chromatin was clarified by centrifugation at 16,000 g for 10 minutes at 4°C. The supernatant was collected and glycerol was added to a final concentration of 5% prior to snap freezing on dry ice and storing at −80°C.
9
Sequential Chromatin Immunoprecipitation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies recognising H3K122ac and H3K64ac were previously described7 (link),9 (link). mESCs were cross-linked in 1% formaldehyde for 10 min and then quenched by the addition of glycine to a final concentration of 0.125 M. Chromatin was sheared using a biorupter (Diagenode) to an average fragment length of ~100 – 200bp. Sequential ChIP was performed as described previously32 (link). Briefly, 5 µg antibodies against H3K4me3 (07-473, Millipore) and H3K27me3 (07-449, Millipore) were covalently coupled to Dynabeads using Invitrogen antibody coupling kit (Cat. 14311D) according to the manufacturer’s instructions. The first ChIP was performed using either H3K4me3 or H4K27me3 antibodies, and the immunoprecipitated chromatin was then eluted with 10 mM DTT, diluted 30 times with RIPA buffer (1X PBS, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, *Roche Protease Inhibitor Cocktail) before performing the second ChIP with anti-H3K122ac9 (link). Purified chromatin was quantified by qPCR using the standard curve method and expressed as % of input bound. Primer details are given in Supplementary Table 2 .
10
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Synchronized D1 adult worms were lysed using a Biorupter (Diagenode) (7 cycles of 30 s on with 30 s pause) at 4oC. Lysate was clarified by centrifugation at 400 x g for 10 min at 4oC. Proteins were transferred from polyacrylamide gels to nitrocellulose membranes (GE Healthcare) at a constant voltage of 75 V, limiting the current to 200 mA for 2 h. Membranes were washed in TBS-T buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) and blocked with 5% skim milk for 1 h at RT. Membranes were incubated with primary antibody in TBS-T with 5% skim milk overnight at 4oC. The blot was then washed 4 times with TBS-T for 10 min each time at RT and incubated with secondary antibody for 1 h at RT. After 4 washes the blot was developed either on an ImageQuant LAS 4000 or ImageQuant 800 system. Images were analyzed in FIJI.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!