148 CHAPTER 5 RNA SEQUENCING Next generation sequencing (NGS) using Illumina’s TruSeq Small RNA Sample Preparation protocol and data filtering were performed as previously described8. Illumina’s TruSeq Small RNA Sample Preparation protocol was used for the generation of cDNA libraries. These libraries were amplified on the flow cells with Illumina’s cluster station (Illumina Inc, San Diego, CA, USA) and sequenced using Illumina’s HiSeq 2000 (Illumina Inc, San Diego, CA, USA). Obtained sequence reads were first quality trimmed, resulting in a >99.9% probability of a correctly identified base of the remaining nucleotides. Secondly, the reads were clipped for adaptor sequences. Thirdly, reads with identical sequences were compiled and counted, resulting in only unique sequences. Finally, each unique sequence was mapped to the reference genome (browser hg19) and only those alignments of at least 18 nucleotides and a maximum of 2 mismatches were retained. Data was visualized on the R2 genomics analysis and visualization platform (http://r2.amc.nl/) and the R2 program was used to generate unsupervised clustering heatmaps using the View Geneset option with 100 most varying genes between the groups as found with the TopLister option, with and log2_z-score transformation settings, as well as sample correlation analyses using the Sample Correlation Map (SCM) option with data as input and log2 transformation setting. Genes with Benjamini and Hochberg p-value ≤ 0.01 were considered differentially expressed.
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