Chromatin isolation by RNA immunoprecipitation (ChIRP) for the long non-coding RNA Pvt1

Long non-coding RNAs (lncRNAs) are emerging as important players in the regulation of several aspects of cellular biology. For a better comprehension of their function it is fundamental to determine their expression in single cells, to identify their subcellular localization and eventually DNA interacting regions. In fact, lncRNAs present a cell-type specific expression and different functions depending on their subcellular localization. C2C12 cells are a model to study muscle pathophysiology and differentiation. We used this model to evaluate Pvt1-DNA interacting regions. We demonstrated that the lncRNA Pvt1, early activated during muscle atrophy, regulate the transcription of DNA and influence mitochondrial respiration and morphology ultimately impinging mito/autophagy and myofiber size in vivo. We evidenced that the long non-coding RNA Pvt1 is highly expressed during skeletal muscle atrophy. Moreover, it is preferentially expressed in fast contracting myofibers. By using high throughput techniques and Fluorescent In Situ Hybridization we evidenced that Pvt1 localize inside the nucleai of myofibers and C2C12 cells. C2C12 cells are widely used as in vitro model to study different aspects of muscle biology such as development, differentiation and metabolism. Because Pvt1 nuclear localization we checked its capacity to interact with DNA by Chromatin isolation by RNA purification (ChIRP). Overall design: We performed the Pvt1 precipitation on C2C12 cells, crosslinked with glutaraldehyde, using 18 biotinilated oligonucleotides spanned along the entaire Pvt1 RNA sequence. Before precipitation we fragmented DNA by sonication at the dimension of ~50-130 bp. We subdivided biotinilated oligonucleotides in odd and even probes to perform two different experimental replicates. Replicates were also performed in two different biological samples. Precipitated samples were processed for RNA and DNA extraction to confirm the enrichment in Pvt1 RNA in the precipitated sample and to evaluate DNA associated fragments. Sequencing runs were performed on PGM Ion torrent producing libraries according to Accel-NGS DNA library kit (Swift Bioscience). Libraries were quantized using the Lib Quant Kit Ion/Rox Low (Roches) and used in the Ion One touch (Thermo Fisher Scientific) for the emulsion PCR as described by the manufacturer. Produced sequences were processed by the Ion Torrent server for the adaptors trimming and their alignement against mouse genome (mm10). Alignement was performed using The Torrent Mapping Alignment Program (TMAP) with default parameters. The software MACS with default settings (macs2 version 2.1.0) was used to identify Pvt1-DNA interaction regions. As backgroud for the peacks identification was used unprecipitated DNA. Unprecipitated DNA was fragmented by Bioruptor UCD-200. having the average dimension of 400 bp. The binding effect were evaluated on the genes that have a peak in the region 2000 bp upstream of the TSS.