We performed chromatin immunoprecipitation (ChIP) using the Magna ChIP G tissue kit (Millipore; catalog #17-20000), according to the manufacturer’s instructions. Briefly, fresh DRG tissues were rapidly removed from anesthetized rats and were stabilized for 3 min using stabilization buffer. Then, the DRGs were incubated in 2% formaldehyde for 20 min at ~26 °C. After being washed three times with PBS, the DRGs were incubated in lysis buffer for 15 min on ice. Finally, the DRG tissues were sonicated (30 s on and 30 s off, repeated 200 times) in ChIP dilution buffer using a water bath sonicator (Qsonica, Newtown, CT) at 4 °C. The sonicated DRG samples were centrifuged at 12,000 rpm for 10 min at 4 °C. Chromatin was pulled down using the following antibodies: IgG (as a negative control; catalog #ab124055, Abcam), total H3 (catalog #2650s, Cell Signaling Technology), H3K9me2 (catalog #ab1220, Abcam), H3K9ac (catalog #39917, Active Motif), H3K27me3 (catalog #9773s, Cell Signaling Technology), and H3K4me3 (catalog #9751s, Cell Signaling Technology). After chromatin precipitation, we performed quantitative PCR using the primers described in Supplementary Table 4 . Data were analyzed and corrected by input and total H3 conditions 60 (link).
Water bath sonicator
Manufactured by Qsonica
The Qsonica water bath sonicator is a laboratory instrument designed for sample preparation. It utilizes ultrasonic energy to agitate samples immersed in a water bath, facilitating processes such as dissolution, dispersion, and extraction.
Automatically generated - may contain errors
Lab products found in correlation
Pcr purification kit, by Qiagen (3 mentions)
Ab124055, by Abcam (2 mentions)
Total h3, by Cell Signaling Technology (2 mentions)
H3k9ac, by Active Motif (2 mentions)
Magna chip g tissue kit, by Merck Group (2 mentions)
H3k27me3, by Cell Signaling Technology (2 mentions)
H3k4me3, by Cell Signaling Technology (2 mentions)
H3k9me2, by Abcam (2 mentions)
Dynapro nanostar, by Wyatt Technology (2 mentions)
Irdye 680rd donkey anti mouse igg, by LI COR (2 mentions)
Intercept tbs blocking buffer, by LI COR (2 mentions)
Irdye 800cw donkey anti rabbit igg, by LI COR (2 mentions)
Nanoluc, by Promega (2 mentions)
Lipofectamine messengermax, by Thermo Fisher Scientific (2 mentions)
Poly d lysine coated cell culture plate, by Greiner (2 mentions)
Trans blot turbo semi dry system, by Bio-Rad (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Thermomixer, by Eppendorf (1 mentions)
Anti phospho atm ser1981, by Merck Group (1 mentions)
11 protocols using water bath sonicator
1
Chromatin Immunoprecipitation from Rat DRG Tissue
2
Purification and Assembly of αSyn Fibrils
3
Protein Analysis of iPS Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
iPS cells were lysed in SDS lysis buffer containing a protease inhibitor cocktail (Roche). Lysates were incubated at 100°C for 10 minutes, and sonicated using a Qsonica Water Bath Sonicator. Western blots were then performed as previously described [17 (link)]. Antibodies used were as follows: anti-BRCA1 (D9 from Santa Cruz Technologies or Cell Signalling Technology), anti-GFP (Roche), anti-Phospho ATM Ser1981 (Millipore), ATM (Cell Signalling) and anti-HSP90 (Enzo Life Sciences).
4
Preparation and Characterization of Mouse α-Synuclein Fibrils
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant full-length mouse αsyn protein was prepared as previously described and generously supplied by Dr. Laura Volpicelli-Daley and Dr. Andrew West [21 (link), 22 (link)]. Before stereotaxic injection in mice, fibrils were generated by incubating purified mouse monomeric αsyn at a concentration of 5 mg/ml in 50 mM Tris (pH 7.4) with 166 mM KCl with constant agitation at 700 rpm at 37 °C for 7 days. Immediately prior to injection, αsyn fibrils were sonicated with a water bath sonicator (QSonica, Newton CT) with 1 s sonication pulses separated by 1 s wait intervals, with every 15 s of sonication separated by 2 min for the total duration of 1 h at A = 30 at 4 °C. For verification of fibril quality, sonicated αsyn fibrils were analyzed by dynamic light scattering (DLS) on a DynaPro NanoStar (Wyatt Technology, Santa Barbara CA) every morning of injection to ensure sonicated fibril average radius was between 20–50 nm prior to injection (Additional file 1 : Figure S1). A subset of unsonicated and sonicated fibrils were examined by transmission electron microscopy (TEM) to confirm fibrillar size and morphology (Additional file 1 : Figure S1).
5
Quantifying NanoLuc protein expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 0.08 million MIA PaCa-2 cells were seeded per well in a 24-well poly-d -lysine-coated cell culture plate (Greiner) and allowed to attach overnight before mRNA transfection with Lipofectamine™ MessengerMAX™ (Life Technologies) according to the manufacturer's protocol. After 24 h incubation, 100 μl of Bolt™ lithium dodecyl sulfate (LDS) sample buffer supplemented with Bolt™ sample reducing agent was added per well of a 24-well plate. The wells were scraped using wide orifice tips and the lysate was transferred into polymerase chain reaction (PCR)-strip tubes and sonicated for 10 × 10 s in a chilled water bath sonicator (QSonica). A 15 μl aliquot of protein extract was separated on 4–12% Bis-Tris plus gels, transferred onto nitrocellulose membranes using the Trans-Blot® Turbo™ semi-dry system (Bio-rad), and blocked for 1 h at room temperature with Intercept™ (TBS) blocking buffer (Li-Cor). Blots were probed with the appropriate primary antibodies overnight at 4°C in blocking buffer supplemented with 0.1% Tween-20, followed by the secondary antibodies IRDye® 680RD donkey anti-mouse IgG or IRDye® 800CW donkey anti-rabbit IgG (Li-Cor) for 1 h at room temperature. Fluorescent signals were imaged and quantified using Odyssey® CLx. Primary antibodies used were: NanoLuc (Promega, N7000) and glyceraldehyde phosphate dehydrogenase (GAPDH; Cell Signaling Technology, #5174)
6
ChIP-seq Library Preparation and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For ChIP-seq, reverse crosslinked DNA treated with RNase A and proteinase K was purified using the Qiagen PCR purification kit. The DNA was then sonicated in a QSonica water bath sonicator (5 min with 15 s ON/OFF pulses, 20% amplitude) to a fragment size of ~200 bp, concentrated and quantified using Qubit dsDNA high sensitivity kit. 1–10 ng of DNA were used to prepare libraries as described previously (Wong et al., 2013 (link)). Libraries were pooled and sequenced on Illumina HiSeq and NextSeq platforms. The raw reads were demultiplexed using Geneious and aligned to the reference genome using bowtie with random assignment of reads to repeats. The mapped reads were normalized to counts per million using Deeptools and visualized in the IGV genome browser. Sequencing data were deposited in the Gene Expression Omnibus (GEO) under the accession number GSE140920.
7
Chromatin Immunoprecipitation from Rat DRG Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed chromatin immunoprecipitation (ChIP) using the Magna ChIP G tissue kit (Millipore; catalog #17-20000), according to the manufacturer’s instructions. Briefly, fresh DRG tissues were rapidly removed from anesthetized rats and were stabilized for 3 min using stabilization buffer. Then, the DRGs were incubated in 2% formaldehyde for 20 min at ~26 °C. After being washed three times with PBS, the DRGs were incubated in lysis buffer for 15 min on ice. Finally, the DRG tissues were sonicated (30 s on and 30 s off, repeated 200 times) in ChIP dilution buffer using a water bath sonicator (Qsonica, Newtown, CT) at 4 °C. The sonicated DRG samples were centrifuged at 12,000 rpm for 10 min at 4 °C. Chromatin was pulled down using the following antibodies: IgG (as a negative control; catalog #ab124055, Abcam), total H3 (catalog #2650s, Cell Signaling Technology), H3K9me2 (catalog #ab1220, Abcam), H3K9ac (catalog #39917, Active Motif), H3K27me3 (catalog #9773s, Cell Signaling Technology), and H3K4me3 (catalog #9751s, Cell Signaling Technology). After chromatin precipitation, we performed quantitative PCR using the primers described in Supplementary Table 4 . Data were analyzed and corrected by input and total H3 conditions 60 (link).
8
ChIP-seq protocol for S. pombe
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For ChIP-seq, immunoprecipitated DNA was cleaned up using the Qiagen PCR Purification Kit after reversing cross-links, RNaseA, and proteinase K treatment. Immunoprecipitated DNA was eluted from column with the provided elution buffer (50 μl × 2), subjected to additional shearing in a Qsonica water bath sonicator at 20% amplitude for 15 min of total shearing time (each cycle is 15 sec on + 15 sec off), followed by vacuum centrifugation to reduce the volume to 30 μl. DNA concentration was measured using Qubit dsDNA HS Kit. One to 10 ng of immunoprecipitated DNA was used for standard Illumina library construction using barcoded adapters and protocol described previously32 (link). Libraries were pooled and sequenced on the Illumina HiSeq2000. Raw reads were mapped to the S. pombe genome using Bowtie’s default parameters, which randomly assigns reads that mapped to repeated regions. Mapped reads were normalized to reads per million (script is available upon request), tiled with igvtools, and visualized with IGV. H3K9me2 and H3K9me3 ChIP-seq were performed twice for wt, clr4Δ, and clr4F449Y cells. All other ChIP-seq experiments were performed once. All ChIP-seq results were verified using ChIP-qPCR.
9
Quantifying NanoLuc protein expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 0.08 million MIA PaCa-2 cells were seeded per well in a 24-well poly-d -lysine-coated cell culture plate (Greiner) and allowed to attach overnight before mRNA transfection with Lipofectamine™ MessengerMAX™ (Life Technologies) according to the manufacturer's protocol. After 24 h incubation, 100 μl of Bolt™ lithium dodecyl sulfate (LDS) sample buffer supplemented with Bolt™ sample reducing agent was added per well of a 24-well plate. The wells were scraped using wide orifice tips and the lysate was transferred into polymerase chain reaction (PCR)-strip tubes and sonicated for 10 × 10 s in a chilled water bath sonicator (QSonica). A 15 μl aliquot of protein extract was separated on 4–12% Bis-Tris plus gels, transferred onto nitrocellulose membranes using the Trans-Blot® Turbo™ semi-dry system (Bio-rad), and blocked for 1 h at room temperature with Intercept™ (TBS) blocking buffer (Li-Cor). Blots were probed with the appropriate primary antibodies overnight at 4°C in blocking buffer supplemented with 0.1% Tween-20, followed by the secondary antibodies IRDye® 680RD donkey anti-mouse IgG or IRDye® 800CW donkey anti-rabbit IgG (Li-Cor) for 1 h at room temperature. Fluorescent signals were imaged and quantified using Odyssey® CLx. Primary antibodies used were: NanoLuc (Promega, N7000) and glyceraldehyde phosphate dehydrogenase (GAPDH; Cell Signaling Technology, #5174)
10
ChIP-seq protocol for S. pombe
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!