Sciclone ngs liquid handler

RNA sequencing (RNA-seq) and quality control (QC) was performed by the Genome Analysis Facility (GAF) of the UMCG. Illumina TrueSeq RNA sample preparation kits were used to generate sequence libraries while using the Perkin Elmer Sciclone NGS Liquid Handler (Waltham, MA, USA). Obtained cDNA fragment libraries were sequenced on an Illumina HiSeq2500 (San Diego, CA, USA) (single reads 1 × 50 bp) in pools of multiple samples. Mus musculus.GRCm38 ensembleRelease 82 reference genome was used to align the trimmed fastQ files with hisat (https://github.com/infphilo/hisat, accessed on 20 March 2017). Sorting of aligned reads was performed using SAMtools (http://www.htslib.org/, accessed on 20 March 2017). The gene level quantification was performed by HTSeq and Ensembl version 82 (https://github.com/htseq/htseq, accessed on 20 March 2017) was used as the gene annotation database. FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 20 March 2017) was used for quality control measurements of raw sequencing data. Picard-tools (https://broadinstitute.github.io/picard/index.html, accessed on 20 March 2017) calculated quality control metrics for aligned reads. Sequencing data was provided via a count table, which could be loaded into RNA-seq analysis software.

RNA isolation, sample preparation, and sequencing was conducted at the University Medical Center Groningen in Groningen, the Netherlands. Each sample was homogenized with zirconia/silica beads in the BeadBeater machine (BioSpec products, Inc.). After homogenization, total RNA was extracted and purified using an RNeasy microkit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. An initial quality check of the samples by capillary electrophoresis and RNA quantification for each sample was performed using the LabChip GX (PerkinElmer, Waltham, Massachusetts, USA). Samples with a minimum amount of 7 ng non-degraded RNA were selected for subsequent sequencing analysis. Sequence libraries were generated using the TruSeq RNA sample preparation kit from Illumina (San Diego, USA) using the Sciclone NGS Liquid Handler (Perkin Elmer). To remove contamination of adapter-duplexes, an extra purification of the libraries was performed with the automated agarose gel separation system Labchip XT (Perkin Elmer). The obtained cDNA fragment libraries were sequenced on an Illumina HiSeq2000 using default parameters (single read 1x100bp) in pools of 10 or 11 samples. Processing of the raw data, including a demultiplexing step, was performed using Casava software (Illumina) with standard settings.

First quality check of and RNA quantification of the samples were performed by capillary electrophoresis using the LabChip GX (PerkinElmer, Waltham, MA, USA). Nondegraded RNA samples were selected for subsequent sequencing analysis. Sequence libraries were generated using the NEXTflex Rapid Illumina Directional RNA‐Seq Library Prep Kit (Bioo Scientific, Austin, TX, USA) using the Sciclone NGS Liquid Handler (PerkinElmer). The obtained cDNA fragment libraries were sequenced on an Illumina Nextseq500 (Illumina, San Diego, CA, USA) using default parameters (single read 1 × 75 bp) in pools of multiple samples, producing on average 4 million reads per sample.