Ribo zero gold

Samples with sufficient RNA quality and quantity were sent for whole transcriptome sequencing at The Center for Applied Genomic (HSC, Toronto, Canada). Library preparation was completed using the TruSeq RNA Library Prep Kit v2 (Illumina, San Diego, CA) using the rRNA depletion kit RiboZero Gold (Illumina, San Diego, CA) according to the manufacturer’s specifications. Paired-end sequencing was performed on the Illumina HiSeq 2500 platform to an average of 250 million paired reads. STAR (Dobin et al, 2013 (link)) was used to align the raw sequencing data to genome reference ‘Homo sapiens UCSC hgl9’ (RefSeq and Gencode gene annotations). Fusion events were independently called using 4 fusion callers:

DeFuse (McPherson et al, 2011 )

TopHat (Kim & Salzberg, 2011 (link))

Ericscript (Benelli et al, 2012 (link))

FusionMap (Ge et al, 2011 (link))

Variant calling from RNAseq was completed using Annovar (Wang et al, 2012).

Unless otherwise specified below, RNA sequencing libraries were prepared using RNA that had been rRNA depleted using Ribo-Zero Gold (Illumina) followed by ScriptSeq-v2 (Illumina), and sequenced on an Illumina HiSeq2500 or Illumina NextSeq500 instrument at Rockefeller genomic resource center. RNA-seq libraries for expression profiling of MDA-LM2 cells with shRNA-mediated XRN2, EXOSC10, RBM7 or UPF1 knockdown were generated using the QuantSeq 3’ mRNA-Seq library prep kit fwd (Lexogen) per the manufacturer’s protocol, and sequenced on an Illumina HiSeq4000 at UCSF CAT.

RNA libraries were prepared according to the manufacturer’s instructions for the TruSeq Stranded Total RNA Sample Prep Kit (Illumina, San Diego, CA). The liquid handling Eppendorf (Hamburg, GER) EpMotion 5075 robot was employed for all AMPure bead clean up. Reverse transcription, A-tailing reaction, and adaptor ligation steps were performed manually. Briefly, 1 μg of total RNA was used as input for ribosomal depletion by RiboZero Gold^™ (Illumina) to remove both cytoplasmic and mitochondrial rRNA. First strand cDNA synthesis was performed using SuperScript III reverse transcriptase, Actinomycin D, and random primers. Second strand cDNA was synthesized using dUTP. The stranded cDNA ends were A-tailed and ligated with index adaptors for multiplex sequencing. The adapter-modified DNA fragments were enriched by 15 cycles of PCR using primers included in the Illumina Sample Prep Kit. The concentration and size distribution of the libraries were determined on an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA). A final quantification, using Qubit fluorometry (Invitrogen, Carlsbad, CA), was done to confirm sample concentration. The libraries were sequenced as 101 paired end reads on an Illumina HiSeq 2000.

RNA was isolated using Qiagen’s RNeasy Plus Mini kit following manufacturer’s instructions from experiments done in biological triplicates. Strand-specific RNA libraries were generated from 1 μg of RNA using TruSeq stranded total RNA with Ribo-Zero Gold (Illumina). For details, see supplementary methods. The expression level and fold change of each treatment group was evaluated using Cuffdiff (16 (link)). For MDA-MB-231 cells, genes were considered differentially upregulated with FC>2 and downregulated with FC<0.5 and p<0.001. For Hs578T cells, genes were considered differentially upregulated with FC>1.5 and downregulated with FC<0.66 and p<0.05. GEO accession number GSE100483.