Melanoma plasticity is regulated by mTOR-mediated crosstalk between MITF and TFE3
ATAC-seq performed to identify chromatin accessibility across the genome. CUT&RUN-seq was performed to identify and compare transcription factor and histone mark occupancy across genome of WT and MITF and TFE3 KO cells. Overall design: SKMEL28 MITF-WT and D6 MITF-KO cells were grown in appropriate condiitons. Cells were lysed in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40: Sigma). Transposition was performed directly on nuclei using 25 µl tagmentation reaction mix (Tagment DNA Buffer #15027866, Tagment DNA Enzyme #15027865 from Illumina Tagment DNA kit #20034210). Tagged DNA was subjected to PCR amplification and library indexing, using the NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs #M0451S) with Nextera DNA CD Indexes (Illumina #20015882), according to the following program: 72°C for 5 minutes; 98°C for 30 seconds, 12 cycles of 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute. The PCR product was purified with 1.8 times the volume of Ampure XP beads (Beckman Coulter #A63881). Library quality was assessed using a BioAnalyzer 2100 High Sensitivity DNA Chip (Agilent Technologies). All DNA libraries that exhibited a nucleosome pattern were pooled and processed for 150 bp paired-end sequencing. SK-MEL-28: WT, MITF-KO, MITF/TFE3-KO; A375: WT, TFE3-KO cells were seeded and grown in appropriate conditions per cell line. 200k cells were harvested by cell scraping and Cleavage Under Targets and Release Using Nuclease (CUT and RUN) was performed for analysis utilizing Anti-MITF, Anti-TFE3, Anti-H3K27me3, Anti-H3K27ac, Anti-H3K9ac, Anti-H3K4me.