Computational protocol: Blocking negative effects of senescence in human skin fibroblasts with a plant extract

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Protocol publication

[…] Library preparation and sequencing were performed on an Illumina HighSeq 2000 Platform (GATC Biotech AG; Konstanz, Germany). All analysis steps were done according to the Tuxedo Suite Pipeline. Briefly, Illumina Casava 1.8.2 software was used for base calling. RNA-seq reads were aligned to hg19 genome assembly using TOPHAT Version 2.0.13 with default parameters.Transcripts were assembled in Cufflinks Version 2.1.1 and differentially expressed genes were predicted by Cuffdiff. Visualisation of data was performed in R using the CummRbund package and The Unscrambler X software (Camo). Functional clustering analysis and filtering for the keyword “secreted” (KW-0964) was done using DAVID v6.8. Pathway analysis was realised with Ingenuity pathway analysis software (Qiagen). Pathway enrichment analysis was performed employing ConsensusPathDB, by using the overrepresentation analysis tool. We searched against pathways in all databases with a minimal overlap and a p-value cut-off of 0.0001. [...] Chemotaxis assay with a µ-Slide Chemotaxis (IBIDI) was performed according to the manufacturer’s manual (Application Note 17 and 23), using conditioned medium from SIPS HDFs and PBMCs freshly isolated from human blood by Ficoll gradient centrifugation. Life cell imaging was performed using a DMI6000 CS microscope (Leica) equipped with a heated CO2 chamber (OKOLAB) and an automated stage. During 12 h, one image/2.5 min was taken. For each condition 30 cells were tracked using the manual tracking plugin and analysed with the chemotaxis and migration tool plugin (IBIDI) in ImageJ. SIPS 1201 HDFs were pre-treated for 4 days with 1201 prior to the experiment. During the conditioning of the medium and the subsequent chemotaxis assay, 1201 was not added to the medium. [...] Error bars are presented as mean ± standard deviation. If not mentioned otherwise, statistical significance was evaluated using a two-tailed Student’s t-test and assumption of equal variance. ANOVA was performed with SigmaPlot 12.5 and the data were tested for equal variance and normal distribution (Shapiro–Wilk). Significance levels were denoted as: *P < 0.05, **P < 0.01 and ***P < 0.001. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus