Transcriptomics profiling of the effect of inducible knock-down of the melanocyte inducing transcription factor (MITF) in melanoma cell lines
MITF plays critical role in development and differentiation of melanocytes and in the context of melanoma, as a lineage survival oncogene. Given its crucial role in melanoma biology, it is very difficult to generate complete knock-out (KO) of MITF and in our hands, those that were generated appear to behave differently than the effect observed using siRNA mediated knock-down, possibly indicative of selection. In order to overcome the limitation of the transient effect of siRNA and study the effect of MITF depletion over a longer period of time, we carried out transcriptomic analyses of Doxycycline inducible shMITF knock down after 8 days in 2 melanoma cell lines MeWo (CVCL_0445) and SkMel 28 (CVCL_0526) Overall design: The cell line was a kind gift from Jiri Vachtenheim DOI: 10.1111/jcmm.13506. MeWo and SkMel28 were seeded on 6 cm dish overnight, the next day, fresh media with 1ug/ml Doxycycline were added. Media with or without Doxycycline were changed every other day. After 8 days, the media was aspirate and the plates snap frozen at -80 °C before total RNA extraction using RNeasy Mini Kit (QIAGEN #74106) as per manufacturer protocol. RNA quality were assessed on Bioanalzyer 2100 (Agilent) using Agilent RNA 6000 Nano Kit (#5067-1511). Only samples with RIN values of over 9.5 were subjected to library prep and sequencing using using the Wellcome Trust genomic service, Oxford. QuantSeq Forward kit (LEXOGEN #0.15.96) was used for library prep, using 500 ng starting material to minimize the PCR amplification step. Samples were sequenced on HiSeq 4000 (Illumina) as paired-end, however, only read 1 contain meaningful data.