NextSeq 500 sequencing; RNA-seq of human SH-EP neuroblastoma cell line with SOX11 overexpression as compared to control at 9h timepoint
RNA-seq of human SH-EP neuroblastoma cell line with SOX11 overexpression as compared to control at 9h timepoint
Cell lines were harvested, centrifuged and stored at -80°C. Cells were grown in RPMI1640 medium supplemented with 10% foetal bovine serum, 2 mM L-Glutamine and 100 IU/ml penicillin/streptavidin at 37°C in a 5% CO2 humid atmosphere, supplemented with 10% tetracyclin-free FBS to avoid leakage. For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11. The constructed plasmid was verified by restriction digest and sequenced by Sanger DNA sequencing (GATC). Lenti-X 293T Cells (Takara, cat#632180) were transfected with the regulator vector pLVX-pEF1a-Tet3G (cat#631353 and Lenti-X Packaging Single Shots (VSV-G) (cat#631275) according to the manufacturer's instructions. The supernatant containing the lenti-virus was collected, filtered through a 0.45 µm filter and concentrated using PEG-IT. SH-EP cells were infected with the concentrated virus and 48 hours of incubation thereafter, the transduced cells were selected using 500 µg/ml G418. Individual clones were obtained by limiting dilution. After clonal expansion, the TET protein expression in each clone was checked by immunoblotting using TetR monoclonal antibody (Clone 9G9) (Clontech, cat#631131). In addition, induction of each expressing clone was tested after transduction with the pLVX-TRE3G-Luc control vector. Selected clones were transduced with lentivirus produced as described above from vector pLVX-TRE3G-Zsgreen1-IRES-hSOX11 and subsequently selected with 1 µg/ml puromycin. Each clone was treated with 0.1 ug/ml Doxycycline for 9h for induction of SOX11 expression or left unaffected in case of the control Total RNA was extracted using miRNeasy kit (Qiagen) according to manufacturer's instructions, including on-column DNase treatment, and concentration was determined with the Nanodrop (Thermo Scientific). 250 ng of RNA isolated from cells was used as input for library preparation with the TruSeq Stranded mRNA Sample Prep Kit from Illumina, followed by quality control on the fragment analyser and qPCR quantificaiton of the libraries using the Illumina Kapa Library quantification kit (Ligtcycler 480 qPCR mix Kapa Code KK4854)