| Construction Protocol | Total mRNA was extracted using TRI Reagent (Sigma-Aldrich) according to manufacturers protocol and DNA was digested with DNA-free Kit (Applied Biosystems). RNA quality was checked by RT-qPCR (as above) and on a 2100 Bioanalyser (Agilent) using a Eukaryote Total RNA Nano Chip (version 2.6). All samples had a RIN value in the range of 9.8-10. Sequencing libraries were generated from 2ug of total RNA per sample with TruSeq RNA sample preparation Kit v2 (Illumina) according to manufacturers protocol. Size and purity of the libraries were analysed on a Bioanalyser High Sensitivity DNA chip (Agilent). Libraries were clustered using TruSeq Single-End Cluster Kit v5-CS-GA (Illumina). Cells were cultured in RPMI 1640 (Gibco), supplemented with 10% fetal bovine serum (FBS) (Gibco), 2mM L-glutamine (Gibco) and 1% Penicillin-Streptomycin solution (Gibco) Azak was dissolved in DMSO in order to apply it to the cells. Therefore a vehicle control consisting of SY5Y-MYCN cells treated with 24h 1µl/ml DMSO only was used in duplicates. Doxycycline (Sigma) dissolved in water was used at a final concentration of 1μg/ml to induce MYCN expression in SY5Y-MYCN. A co-treatment study with Dox and Azak was conducted. SY5Y-MYCN cells were treated with 24h Azak, 24h Azak & 48h Dox and 48h Dox, with biological duplicates. 1 μM RA (dissolved in DMSO) and 1 μg/mL Doxycycline were given individually and in combination. SY5Y-MYCN cells were treated with 24h RA, 24h RA & 48h Dox, and 48h Dox and RNA was extracted in biological duplicates. For the 24h RA & 48h Dox co-treatment cells were treated with Dox for 24h and then with RA and fresh Dox for a further 24h. mRNA was extracted using TRI Reagent (Sigma-Aldrich) according to manufacturers protocol |
