Truseq stranded total rna kit protocol

Illumina’s TruSeq Stranded Total RNA kit protocol was used to generate libraries (see Additional file 1: Supplementary Methods).

Cells were cultured and treated with doxycycline for microprotein expression induction during the specified experimental time periods. Total RNA was isolated with Trizol following manufacturer’s protocol. RNA quantity and purity were measured with the Nanodrop spectrophotometer (Thermo Scientific) and 1μg of total RNA was processed for sequencing analysis. RNA integrity, determined by the RNA integrity number (RIN), was determined with the 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA). Cytoplasmatic and mitochondrial ribosomal RNAs were depleted using the RiboZero Magnetic Gold Kit (Illumina Inc). rRNA-depleted samples were fragmented, cDNA was synthesized and converted into sequentiable libraries using the TruSeq Stranded Total RNA kit protocol (Illumina Inc.). The size and quality of the libraries were assessed with a High Sensitivity DNA Bioanalyzer assay (Agilent Technologies Inc., Santa Clara, CA). Libraries were sequenced in a The NextSeq 500™ (Illumina Inc.), with a read length of 2x76bp. On average, 77 million paired-end reads were generated per sample. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.18.64) and followed by generation of FASTQ sequence files by CASAVA.

The RNA extraction of tumor specimens was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s recommendations. Samples were sequenced at Centro Nacional de Análisis Genómico (CNAG‐CRG, Barcelona, Spain). A modified TruSeq™ Stranded Total RNA kit protocol (Illumina Inc.) was used to prepare the RNA‐seq libraries from samples. Each library was sequenced using TruSeq SBS Kit v3‐HS (Illumina), in paired‐end mode with a read length of 2 × 76 bp. Image analysis, base calling, and base quality scoring of the run were processed by integrated primary analysis software—Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA 1.8.
RNA‐seq reads were aligned to the human (GRCh38/hg38) and mouse (GRCm38/mm10) reference genomes using STAR (version 2.5.1b) and GSNAP (version 2015‐06‐23), respectively, with ENCODE parameters for long RNA. Genes were quantified using RSEM (version 1.2.28) and read counts were used as input for DESeq2 (version 1.10.1). The cut‐off for considering a gene significantly up‐sampled or down‐sampled was FDR < 5%. Subsequently, Gene Set Enrichment Analysis (GSEA) was used to detect coordinated expression within samples using default parameters.