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168 protocols using ChIP-IT High Sensitivity kit

HEK293 cells were treated with or without 2.5 mM PBA in the culture medium for 24 h. After the treatment, cells were fixed with 16% formaldehyde (28906, Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at room temperature. Fixed and fractionated nuclei from HEK293 cells were resuspended in ChIP buffer from ChIP-IT High Sensitivity Kit (Active Motif cat #53040) and sonicated in microtube AFA Fiber Pre-slit Snap-Cap set on Covaris S2 instrument (Covaris, Woburn, MA, USA) at 5% duty cycle, the intensity of 4, and 200 cycles per burst for 16 min. For the immunoprecipitation, ChIP-IT High Sensitivity Kit (#53040, Active Motif, Inc., Carlsbad, CA, USA) and histone H3K27ac antibody (#39133, Active Motif) was used according to the manufacturer’s instructions. After the purification of DNA, the precipitated fraction of DNA was quantified by qPCR by using Fast SYBR Green Master Mix (#4385614, Thermo Fisher Scientific, Waltham, MA, USA) and StepOnePlus™ PCR system (Applied Biosystems). The fold-change of % inputs at the probes to that at GAPDH promoter in each sample was quantified by Ct values of the probes. Primers of the probes are listed in Supplementary Table S2.
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Chromatin was prepared from flash-frozen liver tissue using the Active Motif ChIP-IT High Sensitivity kit (Active Motif), employing a modified protocol described in (Hunter et al., 2019 (link)). All ChIP experiments were conducted with two biological replicates per group, as per ENCODE standards, with replicates handled separately through in vivo to in silico steps.
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Tissue processing and chromatin preparation. Chromatin was prepared from flash-frozen liver tissue using the Active Motif ChIP-IT High Sensitivity kit (Active Motif), employing a modified protocol described in (47) . All ChIP experiments were conducted with two biological replicates per group, as per ENCODE standards, with replicates handled separately through in vivo to in silico steps.
Immunoprecipitation (IP) and DNA elution. 25µg of chromatin (using Nanodrop-measured concentration) was incubated overnight at 4°Cwith a GR antibody cocktail (Protein-Tech 24050-1-A (lot 00044414) and Cell Signaling D8H2 (lot 2) (2µl of each per IP reaction)). As described (47) , to permit direct comparison between samples, and to control for technical variation between ChIP reactions, a spike-in ChIP normalisation strategy was employed (31) (50) . Duplicates were removed with Picard (v2.18.14, Broad Institute). For published ChIP-seq and DNase-seq data, the sratoolkit package (v2.9.2, NCBI) was used to download FASTQ files from the GEO Sequence Read Archive. These were then processed as above.
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Immediately follow sorting, 10,000 cells were pelleted at 4 °C, 1,600 × g for 15 min and re-suspended in SDS lysis buffer. ChIP was conducted based on the manufacturer’s instructions for ChIP-IT High Sensitivity® (HS) Kit (Catalog No. 53040, Active Motif, USA) using anti-H3K4me1 (Abcam, ab8895, 1:100) and anti-H3K27ac (Abcam, ab4729, 1:100) antibodies. ChIP assays were performed in an Bio-Rad CFX96 connect real-time PCR unit using SsoFast™ EvaGreen® qPCR reagent mix (Bio-Rad, 172–5202). Samples were assayed in triplicates and results were normalized to input. Sequences of primer pairs for ChIP–qPCR assays are listed in Supplementary Table 12.
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The cells were seeded onto 10 cm plates and treated with amiodarone (10 μM; Sigma) for 24 h and the ChIP assay was conducted using a ChIP-IT High Sensitivity (HS) kit according to the manufacturer’s instructions (Active Motif, California, USA). Briefly, cells were crosslinked at 37°C for 5 min using 1% formaldehyde. After sonication, the resulting chromatin was diluted 1:10 with ChIP dilution buffer and immunoprecipitated by anti-FoxO1 antibody (Cell Signaling, C29H4) or control IgG. The chromatin-antibody complex was incubated with salmon sperm DNA/Protein A Agarose-50% (Millipore Corp., Billerica, MA, USA) overnight at 4°C with rotation. The DNA was eluted from the beads using ChIP elution buffer and purified by spin column.
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Immediately follow sorting, 10,000 cells were pelleted at 4 °C, 1,600 × g for 15 min and re-suspended in SDS lysis buffer. ChIP was conducted based on the manufacturer’s instructions for ChIP-IT High Sensitivity® (HS) Kit (Catalog No. 53040, Active Motif, USA) using anti-H3K4me1 (Abcam, ab8895, 1:100) and anti-H3K27ac (Abcam, ab4729, 1:100) antibodies. ChIP assays were performed in an Bio-Rad CFX96 connect real-time PCR unit using SsoFast™ EvaGreen® qPCR reagent mix (Bio-Rad, 172–5202). Samples were assayed in triplicates and results were normalized to input. Sequences of primer pairs for ChIP–qPCR assays are listed in Supplementary Table 12.
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Cell nuclei from approximately 20 million formaldehyde crosslinked PC3 cells (1%; 10 minutes at room temperature) were isolated and chromatin was fragmented using sonicator (bioruptor). Lysate were cleared and protein-DNA complexes were isolated using the indicated antibodies and protein-G coated magnetic beads. Chromatin IP was conducted following the standard protocol from ActiveMotif ChIP-IT High Sensitivity® (HS) Kit. Eluted DNA were then subjected to qPCR (SYBR green system) with primers targeting human CCL2 or ATF promoter regions.
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For each sample, cells were trypsinized for 8–10 min, trypsin was quenched with 10 mL of ES media, and 107 cells were obtained. Cells were spun down, washed with 10 mL PBS, fixed for 12 min in a mix of formaldehyde (to a final concentration of 1%) and Fix Buffer (50 mM HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl), and then quenched by glycine (final concentration of 0.125 M). Cells were incubated on ice, and then spun down at 1000g for 5 min. Nuclei were prepared by consecutive washes with Rinse 1 Buffer (50 mM HEPES pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X100), Rinse 2 Buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl), and Shearing Buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris HCl pH 8). After a final spin down, cells were resuspended in 100 μL of Shearing Buffer and 1× protease inhibitor cocktail (Calbiochem) nanodroplets (generous gift from Samantha Pattenden),40 (link) and sonicated in an E110 Covaris Sonicator for 4 min or until DNA was sheared to between 70bp and 500bp (as confirmed by agarose gel).
After sonication, the rest of the protocol was performed with a ChIP-IT High Sensitivity Kit (Active Motif, 53040), and an H3K27ac antibody was used for the pull down (Abcam, ab4729).
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Input and ChIP-DNA analyses were performed using the ChIP-IT High Sensitivity Kit (Active Motif) on uteri from 6-week-old mice. The model-based analysis of ChIP-Seq (MACS) peak-finding algorithm was used to normalize ChIP against the input control (53 (link)). ChIP assays were performed as described previously (33 ). For each ChIP reaction, 100 μg of chromatin was immunoprecipitated using 4 μg of antibodies against HDAC3 (sc11417, Santa Cruz Biotechnology), p300 (sc585, Santa Cruz Biotechnology), PCAF (sc13124, Santa Cruz Biotechnology), and TIP60 (sc25378, Santa Cruz Biotechnology). The sequences of the primers used for HDAC3 response element in the COL1A1 gene were 5’-GAGATGGCATCCCTGGAC-3’ and 5’-CCCATTGGACCTGAACCG-3’, and for HDAC3 response element in the COL1A2 gene, they were 5’-CTGGACTTCCTG-GCTTCAA-3’ and 5’-AGTTCACCCTTGGGACCAG-3’. Immuno-precipitation with normal rabbit IgG was performed as a negative control. The resulting signals were normalized to input DNA.
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ChIP assays were performed with ChIP-IT High Sensitivity® kit (Active Motif; CA). Briefly, microglial cells (4~5 million) isolated from aged male and female mice were cultured under normoxia or OGD/reperfusion treatment conditions; then, cells were harvested and fixed with fixation buffer (containing 1% formaldehyde). Cross-linked cells were washed with cold PBS 3 times and then resuspended in ChIP buffer supplemented with PIC (Protease Inhibitor Cocktail; Active Motif; CA) and PMSF (Phenylmethylsulfonyl Fluoride; Active Motif; CA). Next, the cells were homogenized and the chromatin was sheared to 100~500bp fragments by sonication. A 500-uL volume (100 ug chromatin mixture) was used per immunoprecipitation, and 5 μL (1% of total) was kept as the input DNA. ChIP was carried out with 5 μL of antibodies (anti-H3K4me1, -H3K4me3, -H3K27me1, -H3K27me3; Active Motif; CA) and IgG (Santa Cruz; CA).
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