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The underlying mechanism by which SFRS8 exerted carcinogenesis and its potential targets in MM

ABSTRACT: An RIP experiment was performed in ARP1 cell following the instructions of the article.~5-20 × 10e6 cells were harvested and lysed. Protein A/G MagBeads pre-coated with 5μg of the antibody of interest (SFRS8, abcam) and incubated with cell lysate supernatant overnight at 4°C.The beads containing immunoprecipitated RNA-protein complex were treated with 150μL of Proteinase K buffer to digest the proteins. Specific binding RNAs were isolated by using TRIzol

ORGANISM(S): Homo sapiens

PROVIDER: GSE174823 | GEO | 2022/12/31

REPOSITORIES: GEO

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Explored the underlying mechanism by which ILF3 exerted carcinogenesis and its potential targets in MM

Project description:An RIP experiment was performed in ARP1 cell following the instructions of the article. ~5-20 × 10^6 cells were harvested and lysed. Protein A/G MagBeads pre-coated with 5μg of the antibody of interest (HNRNPA2B1/ILF3, Proteintech) and incubated with cell lysate supernatant overnight at 4°C.The beads containing immunoprecipitated RNA-protein complex were treated with 150μL of Proteinase K buffer to digest the proteins. Specific binding RNAs were isolated by using TRIzol.

2021-11-30 | GSE162303 | GEO

ChIP-seq analysis of Etv2 induced MEF reprogramming and EB differentiation.

Project description:ChIP-seq analysis of Etv2, Brg1 and H3K27ac post Etv2 induction in MEF and ES/EB. Methods: EB or MEF cells were harvested as described above. ChIP assays for ETV2 and H3K27ac were performed from 2 x 10e6 MEFs or 1 x 10e7 EB cells, respectively. Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and the reaction was quenched by glycine at a final concentration of 0.125 M. For the BRG1 ChIP-seq, cells were first crosslinked in 2 mM disuccinimidyl glutarate (DSG; Life Technologies: Cat. #20593) for 30 min then in 1% formaldehyde for 10 min, quenched with glycine for 5 min. The remainder of the ChIP protocol was performed following the protocol described by Magli et al19. We used antibodies against H3K27ac (Abcam, ab4729), ETV2 (Abcam, ab181847), and BRG1 (Abcam ab110641) for the respective pulldowns. For both of the pulldowns, protein A Dynabeads were added to the ChIP reactions and incubated for 30 minutes at room temperature. Magnetic beads were washed and chromatin was eluted. After crosslinking reversal, RNase A, and proteinase K treatment, ChIP DNA was extracted with the Min-Elute PCR purification kit (Qiagen). ChIP DNA was quantified with Quant-it PicoGreen dsDNA Assay Kit (Life Technologies). Sequencing libraries were prepared using equal amount of ChIP DNA per sample as per instructions of SMARTer® ThruPLEX® DNA-Seq Kit (Takara R400675) and SMARTer® DNA Unique Dual Index Kit - 24U Set A (Takara R400665). Sequencing was performed at the University of Minnesota Genome Center with the The NextSeq 550 high-throughput benchtop sequencer.

2022-01-12 | GSE168521 | GEO

Stimulated Human Platelet Lysate: control, PAR1 and PAR4 stimulated

Project description:LC-MS/MS analysis of Control, PAR1-stimulated and PAR4-stimulated platelet lysate digests. Control lysate digest peptides were labeled with "light", PAR1 lysate digest peptides were labeled with "intermediate" and PAR4 lysate digest peptides were labeled with "high" dimethyl labeled. Labeled digests were mixed in a 1:1:1 ratio, separated by SCX and the resulting (phosphopeptide-enriched) peptide fractions were analyzed by LC-MS/MS.

2016-05-11 | PXD000228 | Pride

m6A profiles in shCTRL, shMETTL3 HeLa cells

Project description:Cells were washed with cold PBS twice and scraped from the plates, then centrifuged at 1000 rpm for 5 min to pellet cells. Washed cells were lysed with 400 L of cell lysis buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 0.15% NP-40 and proteinase inhibitor cocktail) and incubated on ice for 10 min. The suspension was carefully add at the top of sucrose buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 24% sucrose), and centrifuged at 3200g for 10 min to collect nuclear pellets. Pellets were resuspended in 250 L of Glycerol buffer (20 mM Tris-HCl pH7.5, 75 mM NaCl, 0.5 mM EDTA pH8.0, 50% glycerol), then then immediately add 250 μl Nuclear lysis buffer (10 mM Tris-HCl pH7.5, 300 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA pH8.0, 1% NP-40, 1M urea). Mix by vortexing for 4 s and incubate on ice for 2 min. Centrifuge the lysate at 13,000 × g for 2 min to precipitate the chromatin–RNA complex. Brieﬂy rinse the chromatin pellets with PBS-EDTA. RNA was extracted with Trizol reagent. RNA libraries were prepared according to manual of SMARTer smRNA-Seq Kit for Illumina (Clontech, 635031).

2019-10-15 | GSE134103 | GEO

Project description:ARP1 deletion sequencing

| PRJNA657114 | ENA

Project description:ARP1 deletion sequencing

| PRJNA656677 | ENA

SIK2-dependent Phosphoproteome and Interactome upon Salmonella infection

Project description:HeLa cells transfected with non-targeting control- and anti-SIK2 siRNA were left uninfected or infected with Salmonella and lysed 30 min pi. Following tryptic-digest peptides were labeled using TMT-pro 16-plex and phosphopeptides enriched. The endogenously tagged SIK2-HA IP workflow. Hela wild type or endogenous SIK2-HA-tagged HeLa cells were left uninfected or infected with Salmonella and lysed 1 h post-infection, followed by HA-IP, Tryptic digest, TMT-labeling and LC-MS/MS analysis.

2021-05-06 | PXD021859 | Pride

RIP-sequence of IGF2BP3 in Ishikawa cells

Project description:To explain the possible molecular mechanism underlying the oncogenic roles of IGF2BP3 in EC, we employed RNA immunoprecipitation (RIP) assays to identify the lncRNAs involved in the regulation of IGF2BP3 function. RIP experiments, high-throughput sequencing and data analysis were performed by Seqhealth Tech (Wuhan, China). RIP assays were carried out on Ishikawa cells. The cells were lysed, and the lysis samples for immunoprecipitation reactions were incubated with anti-IGF2BP3 antibody (ab177477, Abcam, USA) or rabbit IgG (Cell Signaling Technology). The library products were enriched, quantified and finally sequenced on the Illumina PE150 platform.One hundred ninety-one candidates as IGF2BP3-interacting lncRNAs were identified in the RIP-seq results.

2022-03-16 | GSE198620 | GEO

Loss of Asxl2 leads to myeloid malignancies in mice. [ChIP-Seq]

Project description:LK cells of each mouse genotype were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by the addition of lysis buffer, followed by shearing with Bioruptor Pico with water cooler (Diagenode, Seraing, Belgium). The DNA was sheared to an average length of 300-500 bp. Genomic DNA regions of interest were isolated using antibodies against H3K27ac (Diagenode, C15410196), H3K4me1 (C15410194), and H3K4me2 (Abcam, ab32356). Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. After the measurement of DNA concentration with Qubit3.0, the libraries were prepared and sequenced on an Illumina NextSeq 500.

2018-03-08 | GSE97020 | GEO

Autologous Dendritic Cells Loaded with Allogenic Tumor Lysate for Surgically Resected Pancreatic Cancer Patients. Rationale and Clinical Results of a Phase II Study (REACtiVe Trial)

Project description:Determination of shared antigens in Pancreatic ductal adenocarcinoma (PDAC) tumor lysate and the allogeneic tumor drug product. The list of tumor antigenes was thereby dervied from preceeding RNA and literature research (e.g. [Cheevers et al 2009](https://europepmc.org/article/MED/19723653)).

2022-08-17 | PXD025210 | Pride

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