Formaldehyde-fixed cell pellets were snap-frozen in liquid nitrogen and stored at −80 °C before the ChIP assay. Frozen cells were lysed in 120 µl of lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1 mM phenyl methane sulfonylfluoride, 20 mg/ml sodium butyrate, proteinase inhibitor cocktail; Sigma), and chromatin was sheared by sonication, using a BioRuptor (Diagenode), to generate 100- to 500-bp DNA fragments. Chromatin was diluted in 1 ml of radioimmunoprecipitation assay (RIPA) buffer and immunoprecipitation was done overnight at 4 °C by incubating 1 µl of H3K27ac antibody (rabbit polyclonal IgG, lot# Gr167929-1, Abcam) pre-coated on 10 µl protein A–coated magnetic beads (Invitrogen). Immunocomplexes were captured and washed, and ChIP DNA was eluted as described previously9 (link). 1 ng of ChIP DNA was PCR amplified for 18–22 cycles using whole-genome amplification (WGA) primers (WGA-SEQX, Sigma) following the manufacturer’s instructions. Approximately 100 ng amplified DNA was used for preparing a standard Illumina sequencing library (TruSeq Nano DNA HT Sample Preparation Kit, Illumina). Libraries were sequenced on the HiSeq2500 Illumina platform to obtain 50-bp single-end reads (TruSeq Rapid Kit, Illumina). Samples that failed quality control steps, as previously described9 (link), were eliminated from further downstream steps and analysis.
Protein a coated magnetic beads
Manufactured by Thermo Fisher Scientific
Protein A-coated magnetic beads are a type of laboratory equipment used for the purification and isolation of immunoglobulins (antibodies) from complex biological samples. The beads are superparamagnetic and coated with recombinant Protein A, which has a high affinity for the Fc region of antibodies. This allows for the efficient capture and separation of antibodies from other proteins and molecules in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Bioruptor, by Diagenode (2 mentions)
Wga seqx, by Merck Group (2 mentions)
Rnaeasy mini kit, by Qiagen (2 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
H3k27ac antibody, by Abcam (1 mentions)
Truseq rapid kit, by Illumina (1 mentions)
Truseq nano dna ht sample preparation kit, by Illumina (1 mentions)
Bioruptor pico, by Diagenode (1 mentions)
Proteinase k solution, by Qiagen (1 mentions)
Anti h3k27ac antibody, by Abcam (1 mentions)
Affinity column, by Zymo Research (1 mentions)
Fugene 6 transfection reagent, by Promega (1 mentions)
Protease inhibitors cocktail, by Roche (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Smarter ultra low input rna for sequencing kit, by Illumina (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Rnaeasy micro kit, by Qiagen (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
10 protocols using protein a coated magnetic beads
1
Chromatin Immunoprecipitation Sequencing Protocol
2
Protein A-ICAM-1 Binding Assay
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Protein A coated magnetic beads (Invitrogen) were incubated for 1 h with a saturating amount of rmICAM-1-Fc (10 µg/50 µl of beads), washed with PBS containing 0.05% Tween-20 (PBS-T), and then cross-linked with 5 mM BS3 (Thermo Fisher Scientific). Beads were washed with 1 M glycine (pH 2.8) followed by PBS. Beads were then incubated overnight with rmICAM-1-Fc (10 µg), a non-chimeric form of rmICAM-1 (Stemcell Technologies; 10 µg)59 , or cell lysates (300 µg of protein in RIPA buffer) and washed with PBS-T. Molecules bound to beads were eluted by suspending beads in 50 µl of Laemmli sample buffer containing TCEP (50 mM), and by heating (95 °C) samples for 5 min. Proteins within pulled-out fractions were separated using 10% SDS-PAGE gels (10 µl/gel) and ICAM-1 was detected via western blotting4 (link).
3
ChIP-qPCR Assay for RNAPIII Binding
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RNAPIII binding at specific genomic loci was assayed using available ChIP protocols with minor modifications47 (link)48 (link). Briefly, cultured cells were treated as described and immediately fixed in 1% paraformaldehyde, washed in phosphate-buffered saline (PBS), lysed and then sonicated (Bioruptor Pico, Diagenode) to shear DNA to 200–500 bp fragments. Sheared, cross-linked DNA was incubated with 5 μl RNAPIII antibody (RPC32 subunit, #sc-21754, Santa Cruz Biotechnology) and 25 μl protein-A coated magnetic beads (Invitrogen) overnight, washed sequentially in low salt, high salt, LiCl and tris-EDTA (TE) buffers, and then incubated for 2 h at 65 °C in TE buffer containing 1% SDS and proteinase K solution (Qiagen) to reverse crosslinks. Following magnetic removal of protein-A coated beads, extracted DNA was then purified (Qiagen DNA Mini Spin Column), and RNAPIII binding levels at the Fos gene promoter were assayed via qPCR as described above. Ct values for IP samples were normalized to unprocessed (input) DNA, which was not incubated with RNAPIII antibody.
4
ChIP-Seq protocol for H3K27ac analysis
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ChIP was performed as previously described (Seumois et al., 2014 (link)). In brief, cells were fixed with 1% formaldehyde, washed, snap frozen, and stored at −80°C. Frozen samples were lysed and chromatin was sheared by sonication, using a Bioruptor (Diagenode) to yield 100–500 bp fragments. Chromatin was diluted, incubated overnight with protein A–coated magnetic beads (Invitrogen) precoated with anti-H3K27ac antibody (Abcam; Ab4729). Beads underwent a series of washes. Chromatin was then eluted, treated with RNase and proteinase K, and purified by affinity column (Zymo Research). Whole-genome amplification of DNA was performed using WGA-SEQX (Sigma-Aldrich). Sequencing library was prepared and the samples were sequenced using Illumina Hi-Seq. Sequencing data were processed, mapped, and analyzed as previously described (Seumois et al., 2014 (link)). Data have been deposited in the Gene Expression Omnibus database under accession no. GSE74257 .
5
HEK 293 Cell Immunoprecipitation Protocol
6
Chromatin Immunoprecipitation of PU.1 in B Cells
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Cultured B cells stimulated for 4 d with LPS (5 µg/ml) and IL-4 (10 ng/ml) were treated with 2% formaldehyde to cross-link the DNA and protein and incubated for 5 min at room temperature. This reaction was quenched by adding glycine to a final concentration of 125 mM. Cells were then permeabilized in 5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% NP-40, and protease inhibitor cocktail (Roche) for 15 min at 4° C. Lysis buffer consisting of 1% SDS, 10 mM EDTA, and 50 mM Tris-HCl was applied to the permeabilized cells before fragmentation using a Diagenode Bioraptor UCD-200 sonicator.
Fragmented chromatin was diluted to 50 µg/ml in ChIP buffer, containing 0.01% SDS, 1.1% Triton-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, 167 mM NaCl, and protease inhibitors cocktail (Roche). 1 ml was used per immunoprecipitation reaction and 100 µl was used as input control. The chromatin was incubated with either 2.0 µg of rabbit α-PU.1 (T-21 clone; Santa Cruz Biotechnology, Inc.) or the same amount of IgG isotype control overnight and precipitated using 50 µl protein A-coated magnetic beads (Invitrogen). Precipitates were then reverse cross-linked, mRNA and proteins were digested using RNase A and proteinase K by incubating at 37° C for 1 h and at 65° C overnight. After phenol/chloroform extraction, DNA was precipitated using isopropanol and resuspended in TE.
Fragmented chromatin was diluted to 50 µg/ml in ChIP buffer, containing 0.01% SDS, 1.1% Triton-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, 167 mM NaCl, and protease inhibitors cocktail (Roche). 1 ml was used per immunoprecipitation reaction and 100 µl was used as input control. The chromatin was incubated with either 2.0 µg of rabbit α-PU.1 (T-21 clone; Santa Cruz Biotechnology, Inc.) or the same amount of IgG isotype control overnight and precipitated using 50 µl protein A-coated magnetic beads (Invitrogen). Precipitates were then reverse cross-linked, mRNA and proteins were digested using RNase A and proteinase K by incubating at 37
7
Profiling Stress-Responsive Neuronal Populations
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PhosphoTrap profiling experiments were performed according to Knight et al., 2012 (link). Briefly, mice were separated into groups of 4–6 mice per group, restraint stressed for 1 hr and sacrificed following the stress. Naive mice were kept in their home cage until euthanasia. After euthanasia, brains were removed, and septal area were dissected on ice and pooled (4–6 brain samples per experiment replicate). Tissue was homogenized and clarified by centrifugation. Ribosomes were immunoprecipitated using 4 μg of polyclonal antibodies against pS6 (Invitrogen, #44–923G) previously conjugated to Protein A-coated magnetic beads (Thermofisher). A small amount of tissue RNA was saved before the immunoprecipitation (Input) and both input and immunoprecipitated RNA (IP) were then purified using RNAeasy Mini kit (QIAGEN, Germantown, MD) and RNA quality was checked using a RNA PicoChip on a bioanalyzer. RIN values >7 were used. Experiments were performed in triplicates for each group. cDNA was amplified using SMARTer Ultralow Input RNA for Illumina Sequencing Kit and sequenced on an Illumina HiSeq2500 platform. All neurons identified using PhosphoTRAP were validated using qPCR, in situ hybridization, and functional testing.
8
Phospho-TRAP Profiling of Insular Cortex
9
Profiling Active Neuronal Transcriptome
10
PARP1 Protein Immunoprecipitation and Detection
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LNCaP cells were plated in 10% FBS medium and allowed to adhere overnight. The following day medium was replaced with one supplemented with 10% CSS and either DMSO vehicle or 1 μM PDDX. Cells were harvested and cellular extracts supplemented with 1 μM PDDX and 3 μM ABT-888 to inhibit residual PARG and PARP activity, diluted four-fold with TBS, and immunoprecipitated with 0.4 μg of PARP antibody overnight at 4 °C on a rolling incubator. Immune complexes were adsorbed on Protein A coated magnetic beads (#88845, Thermo Scientific, Rockford IL), washed 3 times with TBS, and eluted with Laemmli buffer at 37 °C. For the western blots, 6% of eluate were used for PARP1 and 50% eluate for PAR detection. Images were captured using a GE ImageQuant LAS 500.
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