Computational protocol: Genome-wide profiling of Sus scrofa circular RNAs across nine organs and three developmental stages

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Protocol publication

[…] Nine organs, including the heart, liver, spleen, lung, kidney, ovarium, testis, skeletal muscle, and fat, were collected from three male and female Guizhou miniature pigs at postnatal day 240. We also collected longissimus muscle samples from three Guizhou miniature pigs at postnatal days 0 and 30. Total RNA was isolated from each sample by TRIZOL reagent according to the manufacturer's protocols, after which total RNA was pooled by tissue type and age for RNA-seq analysis. Total RNA was reverse-transcribed according to the manufacturer’s instructions. Paired-end reads 100 bp in length were sequenced by an Illumina HiSeq 2500. RNA-seq data were deposited in the Gene Expression Omnibus (accession codes GSE73763).Reads were first mapped to the pig genome by Bowtie2, after which the mapped reads were filtered out by SAMtools. For the unmapped reads, 20 mers were cut off from both ends of each paired-end read, after which the remaining 60 mers were aligned to the pig genome in reverse orientation by Bowtie2. We used the find_circ package, to identify circRNA. The workflow is shown in .In this study, a circRNA was designated as conserved when it was produced from orthologous genes in different species. We extracted pig-to-human and pig-to-mouse orthologous gene tables from BioMart (www.biomart.org). Human and mouse circRNAs were identified by a previous study.,CircRNA expression was quantified by calculating the number of junction spanning reads per million reads of each sample. Junction spanning reads that were less than 5 reads supported were excluded when determining circRNA expression levels. Differentially expressed circRNAs were identified by the chi-square test with P ≤ 0.05 and had fold-change ≥ 2 (or ≤ 0.5) between any two tissues samples.Four-hundred and eleven mature pig miRNA sequences were obtained from miRBase. We used microtar, Miranda, and RNAhybrid to identify circRNA binding sites for miRNAs. To decrease the proportion of false-positive results, intersections predicted by all three programs were treated as the final set of target miRNAs.The circRNA database is composed of a web interface and a SQLite database engine, which is used to store and manage all data. The data processing programs are written in Python (version 3.4.4). The web interface is built by Django (version 1.8.13) a Python Web framework. Google© charts (https://developers.google.com/chart/) and D3.js (https://d3js.org/) were used for interface development. The circRNA database is coded as a Django project and is deployed by uWSGI (https://uwsgi-docs.readthedocs.io). Web services were built using nginx (version 2.10.0), an HTTP and reverse proxy server. […]

Pipeline specifications

Software tools Bowtie2, SAMtools, find_circ, BioMart, MicroTar, MiRanda, RNAhybrid, Google Charts, D3.js
Databases GEO miRBase
Applications Miscellaneous, Genome annotation, RNA-seq analysis
Organisms Sus scrofa, Homo sapiens, Mus musculus
Chemicals Adenosine Triphosphate, Calcium