ChIP assays were performed as previously described.30 (link) In brief, confluent cells were crosslinked with 4% paraformaldehyde (Sigma, 441,244), and the crosslinking was stopped by the addition of glycerin (Sigma, G8898). The cells were then washed with cold PBS and lysed in FA lysis buffer. Sheared chromatin was subjected to immunoprecipitation with a RUNX2 antibody (#8486, Cell Signaling Technology) or immunoglobulin G (IgG; #2729, Cell Signaling Technology) followed by purification using Protein A/G Dynabeads (GE17152104010150, Merck, Kenilworth, NJ, USA). Protein and RNA were then degraded using Proteinase K (100 μg) and RNase A (1 μg), respectively. The purified chromatin DNA was subjected to quantitative real-time-PCR. The data are expressed as fold change relative to IgG with RUNX2 antibody.
Dynabeads protein a g
Manufactured by Merck Group
Sourced in United States
Dynabeads Protein A/G are magnetic beads coated with recombinant Protein A and Protein G. These beads are designed for the efficient capture and purification of antibodies from various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Magnify chip system, by Thermo Fisher Scientific (2 mentions)
Anti smad7, by Merck Group (2 mentions)
Rabbit anti acetyl histone h4 lys12 h4k12ac igg, by Merck Group (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
T4 pnk, by New England Biolabs (2 mentions)
Runx2 antibody, by Cell Signaling Technology (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Glycerin, by Merck Group (1 mentions)
Immunoglobulin g igg, by Cell Signaling Technology (1 mentions)
Anti smek1, by Merck Group (1 mentions)
Anti hdac1, by Santa Cruz Biotechnology (1 mentions)
Anti mbd3, by Cell Signaling Technology (1 mentions)
Anti hdac2, by Santa Cruz Biotechnology (1 mentions)
Acetyl histone h3, by Santa Cruz Biotechnology (1 mentions)
Anti mta1, by Santa Cruz Biotechnology (1 mentions)
Hiscript 1st strand cdna synthesis kit, by Vazyme (1 mentions)
Random hexamer primer, by Thermo Fisher Scientific (1 mentions)
Superscript 3 rt, by Thermo Fisher Scientific (1 mentions)
Nebnext small rna library prep kit, by Illumina (1 mentions)
Anti msi2 antibody, by Abcam (1 mentions)
13 protocols using dynabeads protein a g
1
ChIP-qPCR Assay for RUNX2 Binding
2
ChIP-seq Analysis of Smek1/2 and Mbd3 in NPCs
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For the ChIP assay, NPCs derived from wild-type or Smek1/2 dKO E11.5 forebrain or transfected with 4 μg pUltra-hot-Mbd3-flag or a pUltra-hot-empty vector for Mbd3 gain-of-function experiments and with pLKO3G-shMbd3 or pLKO3G-shScramble for Mbd3 loss-of-function were treated with 1% formaldehyde for 10 min at room temperature and quenched with 0.125 M glycine for ten more minutes at room temperature. Cross-linked chromatin was sonicated to fragment DNA to 200–1,000 base pairs, and then immunoprecipitation was performed with rabbit anti-IgG, anti-Smek1 (Sigma), anti-Mbd3 (Cell Signaling), anti-HDAC1, anti-HDAC2, anti-MTA1, and acetyl histone H3 (Santa Cruz Biotechnology) antibodies overnight at 4°C, followed by incubation with 50 μl of magnetic Protein A/G Dynabeads (EMD Millipore). Abundance of sequences in immunoprecipitates was determined by PCR and normalized as a fold-value relative to input chromatin. Smek ChIP-seq data were analyzed with the MACS online tool, and cis-regulatory sequences were analyzed using the Genomic Regions Enrichment of Annotations Tool (GREAT) interface (http://bejerano.stanford.edu/great/public/html/ ). We also utilized the Intergrative Genomics Viewer (IGV v2.3) to visualize distribution of ChIP-seq–identified peaks in different genomic regions. Primer sets for ChIP-qPCR are listed in S6 Table .
3
RBM14 Interactome Identification in HEK Cells
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RIP experiments were performed in cell extracts isolated from HEK 293 T cells transfected with pcDNA3.1-RP5-998N21.4. Nuclear extracts were immunoprecipitated with 5 µg of an anti-RBM14 antibody (Abcam, England) or isotype-matched control IgG overnight. RNA-protein-antibody complexes were captured using Protein A/G Dynabeads (Merck). RNA was eluted in accordance with the manufacturer’s instructions. cDNA was synthesized from eluted RNA using a HiScript 1st Strand cDNA Synthesis Kit (Vazyme, China) and analyzed by qPCR.
4
Chromatin Immunoprecipitation of Smad7 and H4K12Ac
5
Chromatin Immunoprecipitation of Smad7 and H4K12Ac
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Chromatin was harvested from RAW246.7 cells or tissue samples using the Magnify ChIP system (Invitrogen). Cross-linked, sheared chromatin was used for immunoprecipitation with protein A/G dynabeads coupled with anti-Smad7, rabbit anti-acetyl-histone H4 Lys12 (H4K12Ac) IgG (EMD Millipore, Billerica, MA) or isotype control from a different species (rabbit). Precipitated DNA was reverse cross-linked and amplified by PCR using primers specific for IKK-β promoter (Table 1
6
Profiling RBMX-RNA Interactome in MOLM13 Cells
7
Cross-Linking Immunoprecipitation (CLIP) Protocol
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CLIP was performed as previously reported18 (link) with some modifications. Briefly, the whole cell lysate from cross-linked (twice by 150 mJ per cm2 of 365 nm UV light) PANC-1 cells were isolated and sonicated, followed by treatment with DNase I (0.5 U/μl, 37 °C for 5 min) and RNase TI (0.2 U/μl, 22 °C for 15 min). Pre-washed Dynabeads protein A/G (Millipore) conjugated with 10 μg antibodies against CSTF2, METTL3, or IGF2BP2 were then incubated with the extraction at 4 °C overnight with rotating. After substantial washing of beads, end repair was performed by using T4 PNK (NEB). RNA was then treated with proteinase K (37 °C for 30 min), acidic phenol/chloroform extraction, and ethanol precipitation, and was subsequently used for library construction by using NEBNext small RNA library prep kit (E7330S) and sequenced on Illumina Hiseq4000. For CLIP-qPCR, the input and immunoprecipitated RNA samples were recovered as described above. cDNA was synthesized with SuperScript III RT (Invitrogen) and random hexamer primers (Invitrogen) and subject to qRT-PCR using specific primers shown in Supplementary Table 3 .
8
Immunoprecipitation of MSI2-RNA Complexes
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Z-138 cells were treated with 5uM of GSK-591 and Ro-0812 for 24 h were collected (20 × 106 cells were used per IP reaction) and washed twice with ice-cold PBS. Cells were lysed in ice-cold IP lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl and 0.5% NP40) for 30 min on ice and frozen at −80 °C immediately to aid the lysis. On the day of IP, the lysate was centrifuged to precipitate the debris. Supernatant was collected and incubated with 5 µg of anti-MSI2 antibody (Abcam) or Rabbit IgG (Millipore) overnight at 4 °C. RNA–MSI2 endogenous antibody complexes were pulled down using Dynabeads Protein A/G (Millipore) and washed five times in 100% IP lysis buffer, 70% IP lysis buffer and 30% PBS, 50% IP lysis buffer and 50% PBS, 30% IP lysis buffer and 70% PBS and 100% PBS. RNA was extracted using the phenol–chroloroform method and quantified by qRT–PCR.
9
m6A-seq protocol for PANC-1 cells
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Total RNA from PANC-1 cells was digested with DNase I and then subjected to 2 rounds of RiboMinus (Illumina) treatment to eliminate ribosomal RNAs. The resultant RNA (200 μg) was then fragmented to about 100 nucleotides in length, incubated with 10 μg of anti-m6A antibody (Synaptic Systems, 202003) before cross-linking with 150 mJ/cm2 254-nm UV light. After incubating with Dynabeads protein A/G (Millipore) at 4°C overnight, m6A-modified RNA was treated with T4 PNK (NEB) on beads, followed by proteinase K treatment, acidic phenol/chloroform extraction, and ethanol precipitation. RNA was subsequently used for library construction with NEBNext small RNA library prep kit (E7330S, NEB) and sequenced on Illumina Hiseq4000.
10
Profiling RBMX-RNA Interactome in MOLM13 Cells
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MOLM13 cells overexpressing RBMX-Flag (RBMX-R2) were collected (20 x 106 cells were used per IP reaction) and washed twice with ice-cold PBS. Cells were lysed in ice-cold IP lysis buffer (50mM Tris-HCL pH 7.5; 300mM NaCl and 0.5% NP40) for 30 minutes on ice and frozen down at −80°C immediately to aid the lysis. On the IP day, lysate was spun down to precipitate the debris. Supernatant was collected and incubated with 7.5 ug anti-Flag antibody (Sigma Aldrich clone M2- F1804) or mouse IgG (Millipore 12-371) overnight at 4°C. RNA-RBMX-Flag-antibody complexes were pulled down using Dynabeads Protein A/G (Millipore) and washed 5 times in 100% IP lysis buffer, 70% IP lysis buffer and 30% PBS, 50% IP lysis buffer and 50% PBS, 30% IP lysis buffer and 70% PBS, and 100% PBS. RNA was extracted using phenol-chroloroform method and quantified for qRT-PCR.
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