Illumina HiSeq 3000 paired end sequencing; Single cell RNA-seq of human RPE1-MYCN-ER cells with treatment of EtOH (0h) or tamoxifen for 22h, 23h and 24h to activate MYCN.
Cell lines were harvested, centrifuged and stored at -80°C. Cell lines were harvested, kept on ice before processing. RPE1–MYCN-ER were cultured in DMEM/F-12 HEPES, supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamin and 0,01mg/ml Hygromycin B according to standard procedures. Moreover, 1 ug/ml puromycin was added to the RPE1–MYCN-ER medium composition as a selection marker for the 4-OHT inducible construct. To evaluate transcriptomic changes RPE1–MYCN-ER were plated in T25 iand treated with or without 400 nM 4-OHT (H6278; Sigma-Aldrich) for 22h, 23h and 24h. Control was supplied with the same amount of EtOH as was present in the treated cells and was always ≤0.5% of the total volume. Chromium Single cell 3' reagent kits v3 Per sample, 5000 cells were loaded to end up with 2500 captured cells. During cDNA amplification, 12 PCR cycles were used. To add an index to the samples, another 12 PCR cycles were used. To check the final quality of the generated libraries, the samples were loaded on a Bioanalyzer High sensitivity chip (Agilent Technologies, Santa Clara, CA, US), and the quantity was checked with qPCR according to the Illumina protocol 'Sequencing Library qPCR Quantification protocol guide', version February 2011. Samples were pooled equimolarly and sequenced on a Hiseq4000 lane (Illumina, San Diego, CA, USA), generating paired-end reads of 150 base pairs. Afterward, the data were trimmed to paired-end reads of 28 and 91 base pair.