Transcriptome Profiling Using Illumina HiSeq

Total RNA was extracted using RNeasy Plus Micro Kit (Qiagen #74034) according to the manufacturer’s protocol. RNA quality was checked on Bioanalyzer (Agilent) using RNA 6000 Nano Kit (Agilent #5067-1511). Total RNA samples with RIN value > 7 were used for library preparation. cDNA libraries were prepared using TruSeq RNA Sample Prep v2 (Illumina #RS-122-2001) and subsequent next-generation sequencing was performed on an Illumina HiSeq 4000 platform by Macrogen INC. Sequencing from each sample was performed: 101nt long pair-end reads with at least 80 M reads per sample. Differential expression analysis was performed on the Galaxy platform by the following pipeline: Paired-End sequences obtained from the HiSeq 4000 platform were trimmed using the Trimmomatic v0.36.3 [61 (link)]. Sequence reads that pass quality filters were mapped to the human reference genome hg38 using Bowtie 2 v2.3.4.1 [62 (link)]. Mapped reads were counted using featureCounts v1.6.0.3 [63 (link)] followed by calculating differential gene expression with DESeq2 v2.11.40.1 [64 (link)]. Differentially expressed transcripts were filtered according to the parameters of log2FC ≥ 0.5 for upregulated and log2FC ≤ −0.5 for downregulated genes. Adjusted p-value ≤ 0.01 was considered as significant.