GSM8207783: tum 9; Homo sapiens; RNA-Seq
Thirteen tumour samples stored in RNAlaterTM solution (Invitrogen, Thermo Fisher Scientific) were provided by Dr Akinyele Adisa from the Department of Oral and Maxillofacial Surgery and Oral Pathology, College of Medicine, University of Ibadan, Nigeria. These were selected after histopathological re-evaluation by pathologists (KH and CM) and confirming that over 90% of the tissue represented tumour. Total RNA was extracted from tumours by manual macro-dissection using RNeasy Mini Kit (QIAGEN) following manufacturer's instructions and quantified using a NanoDrop spectrophotometer. A total of  25µl of RNA from each tumour were sent to Novogene (Cambridge, UK) to perform RNA- sequencing. The company performed a quality control of the samples firstly through an Agarose gel electrophoresis to test the RNA degradation and potential contamination; and secondly with Agilent 2100 to check the RNA integrity and quantitation.  First, the mRNA was fragmented randomly by adding fragmentation buffer, then the cDNA was synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H and DNA polymerase I were added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment. Then the quality control of the library consisted of three steps (Qubit, Agilent 2100 and qPCR). The qualified libraries were fed into Illumina sequencers after pooling according to its effective concentration and expected data volume.