Dataset features


Application: RNA-seq analysis, sRNA-seq analysis
Number of samples: 41
Release date: Jun 17 2018
Last update date: Nov 29 2018
Access: Public
Diseases: Dental Caries
Dataset link Cooperation of GRSF1 and the mitochondrial degradosome (hSuv3-PNPase complex) in degradation of mitochondrial RNA

Experimental Protocol

There are 2 sets of data which correspond to two RNAseq studies (degradosome set and GRSF1 set). Each set includes experiments which were performed in triplicates. The aim of the first studies was to analyze the effect of inactivation of the mitochondrial degradosome components (hSuv3, PNPase) on mitochondrial RNA (mtRNA). In the second studies we examined influence of GRSF1 silencing of mtRNA. In both cases we analyzed long transcripts and small RNAs. Strand-specific library preparation procedures were applied. Ligation-based approach was used to analyze short RNAs (from ~20 to ~200 nucleotides) whereas longer RNAs (> ~100 nucleotides) were analyzed after their random fragmentation followed by reverse transcription primed with random oligomers (dUTP-based protocol). RNA was isolated from unfractionated cells using TRI-Reagent. Before preparation of long transcripts libraries total RNA was subjected to rRNA depletion. This step was omitted in preparation of libraries dedicated for analysis of short RNAs. Total RNA was used for preparation of small RNA libraries. Libraries from different studies (degradosome and GRSF1) were sequenced with the help of different Illumina sequencing platforms. Dysfunction of hSuv3 was achieved by inducible expression of its dominant-negative mutant form (hSuv3-G207V), which we shown to be equivalent to hSuv3 silencing (PMID: 19864255), whereas PNPase or GRSF1 were depleted by siRNA transient transfections. In the case of GRSF1 studies as controls we analyzed untreated cells and cells transfected with non-targeting siRNA. In the case of degradosome studies as controls we analyzed untreated cells and cells expressing siRNA-insensitive version of PNPase (rescue experiment).










Tomasz Kulinski