Illumina Genome Analyzer IIx paired end sequencing; MYCN overexpression in SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN cell line
MYCN overexpression in SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN cell line
TOT_1h_B
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) 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 Paired-End Cluster Kit v5-CS-GA (Illumina). Besides the SY5Y-MYCN untreated control (also referred to as 0h), SYSY-MYCN cells were transfected with MYCN through a doxycline (Dox) inducible system. RNA for 5 time points untreated (0h), 1h, 4h, and 24h together, so that all cells had the identical time growing in culture, with MYCN overexpression time being the only variable. To achieve the different overexpression time points the treatment start time was staggered. At time-points 0h, 1h, and 4h the RNA was split in 2 parts. One part was used for total mRNA sequencing. The other one was used to separate 4sU labelled mRNA from 4sU unlabelled with beads. Two biological replicates for every time-point were extracted for the total, labelled, and unlabelled RNA. DTA analysis was used to label freshly produced mRNA for 3 of the time points. 30 minutes before time-points 0h, 1h, and 4h, 4-thiouridine (4sU) was added to the cells. From this time-point on, 4sU is used in the synthesis of mRNA and therefore labels the freshly produced mRNA. After 30 min, all remaining unlabelled mRNAs are products from synthesis before 30 min. The labelled and unlabelled parts combined are called total mRNA which is simply traditional mRNA-sequencing. mRNA was extracted using TRI Reagent (Sigma-Aldrich) according to manufacturers protocol