Sciclone ngsx workstation

RNA was extracted and purified from the Ptm lymphocytes using the RNeasy Mini kit (Qiagen) following manufacturer's protocol. Total RNA integrity was checked using an Agilent 2200 TapeStation (Agilent Technologies) and quantified using a Trinean DropSense96 spectrophotometer (Caliper Life Sciences). RNA-seq libraries were prepared from total RNA using the TruSeq RNA Sample Prep Kit v2 (Illumina) and a Sciclone NGSx Workstation (PerkinElmer). Library size distributions were validated using an Agilent 2200 TapeStation (Agilent Technologies). Additional library QC, blending of pooled indexed libraries, and cluster optimization were performed using Life Technologies’ Invitrogen Qubit 2.0 Fluorometer (Life Technologies-Invitrogen). RNA-seq libraries were pooled (6-plex) onto a flow cell lane. Sequencing was performed using an Illumina HiSeq 2500 in rapid mode employing a paired-end, 50 base read length (PE50) sequencing strategy. Image analysis and base calling were performed using Illumina's Real Time Analysis v1.18 software, followed by 'demultiplexing' of indexed reads and generation of FASTQ files, using Illumina's bcl2fastq Conversion Software v1.8.4.

RNA was sequenced at Northwest Genomics Center, where next-generation sequencing libraries were prepared from 1.25 µg of total RNA in a high-throughput format, using the TruSeq Stranded mRNA kit (Illumina, San Diego, CA, USA). All the steps required for sequence library construction were automated and performed on a Sciclone NGSx Workstation (Perkin Elmer, Waltham, MA, USA). During library construction, rRNA was depleted by means of a poly-A enrichment, and first and second strand cDNA syntheses were performed. Each library was uniquely barcoded using Illumina adapters and amplified by PCR. After amplification and cleanup, library concentrations were quantified using the Quant-it dsDNA assay (Life Technologies, Carlsbad, CA, USA). Final libraries were normalized and pooled based on Agilent 2100 Bioanalyzer results (Agilent Technologies, Santa Clara, CA, USA) and size selected using a Pippin Prep (Sage Science, Beverly, MA, USA). Pooled libraries were diluted to a final concentration of 2–3 nM for sequencing on a HiSeq 4000, to a read depth of 30 million base pairs. Samples were multiplexed and sequenced on a HiSeq 4000. Lane-level sequencing reads were base quality checked using the FASTX-toolkit and FastQC, and aligned to hg19 with a reference transcriptome Ensembl v67, using TopHat2 suite [56 (link)] followed by matefixing, as described previously [28 (link)].

RNA from retinas was isolated using TRIzol (Invitrogen) and total RNA integrity was checked using an Agilent 4200 TapeStation and quantified with a Trinean DropSense96 spectrophotometer. RNA-seq libraries were prepared from total RNA using the TruSeq RNA Sample Prep kit (Illumina) and a Sciclone NGSx Workstation (PerkinElmer). Library size distributions were validated using an Agilent 4200 TapeStation. Additional Library quality control, blending of pooled indexed libraries, and cluster optimization were performed using Life Technologies’ Invitrogen Qubit Fluorometer. RNA-seq libraries were pooled (4-plex) and clustered onto a flow cell lane. Sequencing was performed using an Illumina HiSeq 2500 in rapid mode employing a paired-end, 50 base read length (PE50) sequencing strategy.