Whole Genome Sequencing of Tongue cancer samples and cell line
Whole Genome Sequencing of Tongue cancer samples and cell line
The samples AD2881 and AD2882 are primary tongue tumor and paired adjacent tongue tissue normal, respectively, from a clinically early stage (T2N0M0) tongue cancer patient. The samples were collected from a male patient, aged 38 at the time of sample collection. The patient has a habit of tobacco chewing and alcohol consumption. NT8E cell line was established within the Tata Memorial Centre (Mulherkar et al., Cancer Letters, 1997) and was acquired from the Tata Memorial Hospital, Mumbai, India. NT8E is an epithelial cell line established from the surgical specimen of an untreated, upper aero-digestive tract tumor, diagnosed as a squamous carcinoma of the pyriform fossa. The sex and age of the donor are not available. Also, the NT8E cell line is not deposited in the cell line repositories, such as ATCC, and hence, there is no catalogue number. Each sample was collected by scrapping the cells from a 10cm cell culture dish. Pellet was stored at -20 degrees. NT8E cells were cultured in DMEM medium. The media was supplemented with 10% FBS, 2.5 mg/ml Amphotericin-B (Abbott) and 1.25 μl/ml Gentamycin (Abbott). The cells were cultured at 37 °C in a 5% CO2 incubator. Genomic DNA extraction from the tissue samples was performed using AllPrep DNA/RNA/miRNA Universal Kit (Cat No. 80224, QIAGEN). About 20-30mg tissue sections were cut into small pieces and subjected to bead-based homogenization in 600µL lysis buffer using FastPrep homogenizer (MP Biomedicals, USA) and further processed for genomic DNA extraction as per manufacturer's protocol. Genomic DNA from the cell lines were extracted using DNeasy Blood and tissue DNA extraction kit (Cat No. 69506, QIAGEN) according to manufacturer's instructions. Whole genome sequencing (WGS) library was prepared with Illumina-compatible NEXTFlex Rapid DNA Sequencing Bundle (5144-02, NEXTFlex, BIOO Scientific, Inc. USA). Briefly, 300 ng of Qubit quantified DNA was sheared in Covaris microTUBE AFA (520045, Life Technologies) using Covaris S220 sonicator (4465653, Life Technologies, Covaris, Inc. USA) to generate specific fragments with size range of 300-400 bp. The fragment size distribution was verified on Agilent 2100 Bioanalyzer and subsequently purified using HighPrep magnetic beads (AC-60050, MagBio Genomics, Inc, USA). Purified fragments were end-repaired, adenylated and ligated to Illumina multiplex barcode adaptors as per NEXTFlex Rapid DNA sequencing bundle kit protocol. Adapter-ligated DNA was purified using HighPrep beads. Resultant fragments were amplified for 5 cycles of PCR using Illumina-compatible primers provided in the NEXTflex Rapid DNA sequencing Bundle. Final PCR product (sequencing library) was purified with HighPrep beads, followed by library quality control. Sequencing library was quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA) and its fragment size distribution was analyzed on Agilent 2100 Bioanalyzer (5067-4626, Agilent High Sensitivity DNA Bioanalyzer Kit).