Ribo zero human kit

A BGI SEQ-500 SE50 sequencing library was prepared from total muscle RNA and sequenced at a depth of 60 Mio paired-end fragments on a SEQ-500 machine.³¹ (link) Total RNA was isolated in triplicate from primary fibroblast cell lines using the mirVana miRNA Isolation Kit (Ambion) and DNAse treated with the DNA-free DNA Removal Kit (Ambion). RNA sequencing (RNA-seq) libraries were prepared with Illumina TruSeq Stranded polyA enriched RNA with Ribo-Zero Human kit and were sequenced on an Illumina HiSeq 2500 platform according to paired-end protocol. Control muscle RNA sequences were obtained as described previously.³² (link) The quality of sequencing reads was checked with FastQC. Reads were aligned with the STAR (v2.5.2b) aligner and the two-pass protocol that is outlined in GATK documentation. Number of reads mapped to Ensembl GRCh38 v86 genes was counted with HTSeq-count.³³ (link) Differentially expressed genes between affected and control individuals were identified with Bionconductor package DESeq2.³⁴ (link) Genes with a false-discovery rate ≤ 0.1 and a log2 fold change ≥ 1 were considered differentially expressed. Gene-set enrichment analysis for gene ontology terms was performed with the ConsensusPathDB (CPDB) web tool.

RNAseq libraries were prepared with Illumina TruSeq Stranded polyA–enriched RNA with Ribo-Zero Human kit and sequenced as previously described (Burns et al, 2018 (link)). Exosc3 morphant zebrafish and control zebrafish RNAseq reads were downloaded from European Nucleotide Archive Study: PRJNA470927 (Francois-Moutal et al, 2018 (link)). Raw sequence reads were trimmed to 50 bp with FastX-toolkit v.0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/index.html) and aligned to complete zebrafish (danRer11) or human (hg38) reference genomes, using the STAR aligner v.2.5.3a two-pass protocol (Dobin et al, 2013 (link)). Differential expression analysis was performed with HTSeq v.0.9.1 (Anders et al, 2015 (link)) and DESeq2 v.1.12.4 (Love et al, 2014 (link)). Gene set over-representation analysis was performed via http://cpdb.molgen.mpg.de/. Bedfiles of non-coding RNA types were prepared from gene coordinates available at Ensembl Biomart except for cytosolic tRNAs, where sequences and coordinates were obtained from GtRNAdb (http://gtrnadb.ucsc.edu/). BEDTools v.2.26.0 was used to obtain per gene strand–specific counts of non-coding RNAs. RNA sequencing files were deposited to National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE151452.
RNAseq data from muscle samples of two EXOSC9 patients has been published previously (Burns et al, 2018 (link)).