Computational protocol: Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection

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Protocol publication

[…] To identify possible Smad-dependent transcripts, RVFV and mock infected normalized RNASeq datasets (GEO Submission # GSE102481 []) were utilized for promoter analysis. Average fold changes (RVFV versus mock infected) for each time point from three independent experiments were determined. Those transcripts with Student’s t-test p ≤0.001 and difference of 2-fold or more in expression at 9 and/or 18hpi were considered for analysis. Duplicated Ensembl gene ids (ENSGs) were removed and Ensembl transcript ids (ENSTs) were obtained (Biomart Converter program [http://www.biomart.org/index.html]).Next, ENSTs were submitted to Genomatix Software Suite Gene2Promoter program (http://www.genomatix.de). This program assigns the accession number to a gene locus in Genomatix’ ElDorado database which relies on Homo sapiens GRChr38 build and contains both predicted and experimentally validated promoter sequences. Searches were performed with the following criteria: 1) Homo sapiens promoters were analyzed, 2) vertebrate Smad family matrices were to be applied (V$SMAD), and 3) Smad transcription factor response elements needed to be within 1000bp of a defined transcriptional start site. There are five Smad family matrices, V$SMAD.01, V$GC_SBE.01, V$SMAD3.01, V$SMAD3.02 and V$SMAD4.01 (Matrix Library 9.3 March 2015) with a matrix length varying from 8 to 11 base pairs. The output list was further filtered to focus on those ENSTs that had a Smad matrix similarity of ≥ 0.995 ().ENSGs identifiers and the average fold change values for the 913 transcripts that contained predicted Smad response elements within their promoters were imported into Ingenuity Pathway Analysis (IPA, Qiagen Bioinformatics; https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) to determine which cellular networks are populated by these transcripts. Only 898 could be mapped. A comparative analysis of all datasets was performed using a 1.5-fold cutoff to identify canonical pathways that could be related to RVFV pathology to focus on for chromatin immunoprecipitation. […]

Pipeline specifications

Software tools BioMart, Genomatix Software Suite, Gene2Promoter, IPA
Databases ElDorado
Applications Genome annotation, RNA-seq analysis
Organisms Homo sapiens
Diseases Coccidioidomycosis, Rift Valley Fever
Chemicals Calcium