GSM4861329: 904 2 CTC 2months Nefiracetam; Homo sapiens; RNA-Seq
RNA was extracted using a RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNase-treated RNA was assessed with a NanoDrop1000 (ThermoScientific, Waltham, MA, USA). Total RNA was converted to cDNA and evaluated for RT-PCR array per the manufacturer's instructions (Qiagen, Hilden, Germany). A RNeasy Mini kit (Qiagen) was used to isolate RNA for library preparation (Illumina TruSeq RNA Sample Preparation Kit; San Diego, CA, USA) and sequencing (Illumina HiSeq2000, 50bp paired-end reads, 50 million high-quality sequencing fragments per sample, on average). Data was analyzed by Rosalind (https://rosalind.onramp.bio/), with a HyperScale architecture (OnRamp BioInformatics, Inc., San Diego, CA). Reads were trimmed using cutadapt, quality scores were assessed using FastQC, and reads were aligned to the Homo sapiens genome build hg19 using STAR. Individual sample reads were quantified using HTseq and normalized via Relative Log Expression using DESeq2 R library. DEseq2 was also used to calculate fold changes and p-values and perform optional covariate correction. Gene clustering of differentially expressed genes was done using the Partitioning Around Medoids method using the fpc R library. Functional enrichment analysis of pathways, gene ontology, domain structure, and other ontologies was performed using several databases (HOMER, Interpro, NCBI, MSigDB, REACTOME, WikiPathways).