Computational protocol: The RBM14/CoAA-interacting, long intergenic non-coding RNA Paral1 regulates adipogenesis and coactivates the nuclear receptor PPARγ

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Protocol publication

[…] Total RNA (100–300ng) from 3T3-L1 cells was processed for labelling, purification and hybridized to Affymetrix Genechip Mouse Genome 430 2.0 or to Mouse Transcriptome Array 1.0 according to the manufacturer’s protocol. Raw data (available on the GEO website under the accession number GSE 97241) were pre-processed using the GCCN and SST algorithms (Expression Console, v1.4.1. Affymetrix). RMA background correction and gene-level probe set summarization were performed with the Partek Genomics Suite software (v6.6, Partek Inc.). Microarray analysis of leptin-deficient (ob/ob) mouse WAT has been described elsewhere. Microarray data from mouse WAT fed either a chow or a high fat diet were from the GEO dataset GSE21069 . Human WAT RNAs were analysed as described.Human gene symbols were attributed to each murine gene using the Orthologue Conversion software (https://biodbnet-abcc.ncifcrf.gov/db/dbOrtho.php) and resulting files were analysed with the GSEA software [Broad Institute, v2.2.2]. Pathway-enrichment scores were calculated with the GSEA pre-ranking tool and the KEGG Pathway gene set. Default parameters were used except for the permutation number (10,000) and the enrichment score statistic (weighted). The GEO dataset is available under the number GSE97241. [...] Three softwares were used with default parameters to determine lincRNAs’ coding potential (CPC, http://cpc.cbi.pku.edu.cn/); CPAT, http://lilab.research.bcm.edu/cpat/index.php) and GenView2 (http://bioinfo.itb.cnr.it/~webgene/wwwgene.html). GAPDH and XIST RNA were used as representative of mRNA and lincRNA respectively. Potential ORFs were converted into protein sequences and matching peptides were searched in four protein databases [UniProtKB (SwissProt and TEMBL; http://www.uniprot.org/); PDB (http://www.rcsb.org/pdb/home/home.do); Ensembl (www.ensembl.org/)]. [...] Protein separation, in-gel trypsin cleavage and mass spectrometry analysis were carried out as described. Peptide separation was performed using an EASY-nLC 1000 UHPLC (Thermo Scientific) equipped with a 75 µmX 2 cm Acclaim PepMap 100 pre-column with nanoViper fittings and a 50 µm I.D. × 500mm Acclaim PepMap RSLC analytical column (Thermo Scientific). Peptides were eluted using a 5%-30% acetonitrile gradient for 60 min. at a flow rate of 300 nL/min. The Q-Exactive instrument acquisition mode was set to the top 10 MS2 method. The survey scans were taken at 70,000FWHM (at m/z 400) resolving power in positive mode and using a target of 1e6 and default charge state of + 2. Unassigned and + 1 charge states were rejected, and dynamic exclusion was enabled for 30 sec. The scan range was set to m/z 300–1600 m/z. For MS/MS, a microscan was obtained at 17,500FWHM and with an isolation window of 3.0 m/z, using a scan range between m/z 200–2000 m/z. Tandem mass spectra were processed with the Thermo Scientific Proteome Discoverer software v 1.3. Spectra were searched against UniprotKB/Swiss-Prot mouse databases (version 09/2015) using the SEQUEST HT algorithm (v1.3.0.339). The search was performed choosing trypsin as the cleaving enzyme with one missed cleavage site allowed. Precursor mass tolerance was 10 ppm, and fragment mass tolerance was 0.1 Da. N-terminal acetylation, cysteine carbamidomethylation and methionine oxidation were set as variable modifications. Peptide identification was performed with the Percolator algorithm by selecting only peptides with a q-value < 0.01, which corresponds to a false discovery rate (FDR) of 1%. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet
Databases UniProt UniProtKB
Application MS-based untargeted proteomics
Organisms Homo sapiens, Mus musculus