TruSeq Stranded mRNA

Protocol tips

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	The protocol is optimized for 0.1–1 µg of total RNA

Protocol tips
The protocol is optimized for 0.1–1 µg of total RNA

Publication protocol

Library preparation and RNA-seq were carried out as described in the Illumina TruSeq Stranded mRNA Sample Preparation Guide, the Illumina HiSeq 1000 System User Guide (Illumina, Inc., San Diego, CA, USA), and the KAPA Library Quantification Kit—Illumina/ABI Prism User Guide (Kapa Biosystems, Inc., Woburn, MA, USA). In brief, 300 ng of total RNA was used for purifying the poly-A containing messenger RNA (mRNA) molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented to an average insert size of 200–400 bases using divalent cations under elevated temperature (94 °C for 4 min). The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM), followed by second-strand cDNA synthesis using DNA Polymerase I and RNase H. The incorporation of dUTP in second-strand synthesis quenches the second strand during amplification, because the polymerase used in the assay is not incorporated past this nucleotide. The addition of Actinomycin D to First Stand Synthesis Act D mix (FSA) prevents spurious DNA-dependent synthesis, while allowing RNA-dependent synthesis, improving strand specificity. These cDNA fragments then had the addition of a single “A” base and subsequent ligation of the adapter. The products were purified and enriched with PCR to create the final cDNA library. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit. Equimolar amounts of each library were used for cluster generation on the cBot (TruSeq SR Cluster Kit v3). The sequencing run was performed on a HiSeq 1000 instrument using the indexed, 50 cycles single read (SR) protocol and the TruSeq SBS v3 Kit. Image analysis and base calling resulted in .bcl files, which were converted into .fastq files by the CASAVA1.8.2 software. Library preparation and RNA-seq were performed at the service facility “KFB—Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany).
The RNA express workflow on Illumina BaseSpace was used to determine differential gene expression in two biological replicates of control retinas, light-exposed retinas, and light-exposed retinas treated with minocycline, respectively. In brief, alignment of RNA-seq reads to the mouse UCSC mm10 genome and mapping to genes was performed with STAR aligner [35]. Differential gene expression between the two biological replicates each was calculated with DESeq2 [36]. The cutoff of genes considered to be differentially expressed was a log2 fold change of ≥2 or ≤−2.
Integrative analysis of genome-wide expression activities was performed with the Gene Expression Dynamics Inspector (GEDI), a Matlab (Mathworks, Natick, MA) freeware program which uses self-organizing maps (SOMs) to translate high-dimensional data into a 2D mosaic [37]. Each tile of the mosaic represents an individual SOM cluster and is color-coded to represent high or low expression of the cluster’s genes, thus identifying the underlying pattern.
The RNA-seq raw data and normalized DESeq2 counts of this study are publicly available at the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) as series record GSE71025.

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