TFEB Chromatin Immunoprecipitation Assay

ChIP was performed as previously described^{68 (link)}. Briefly, for each ChIP experiment 8–10 million cells were cross-linked with 1% formaldehyde (Pierce) for 10 min at room temperature, nuclei were isolated and chromatin was sonicated at 4 °C using a Bioruptor (Diagenode) for 25 cycles (30 s ‘ON’ and 30 s ‘OFF’, power setting high). For each immunoprecipitation 18 μl of antibody against TFEB (Cell Signaling, no. 37785, D2O7D) were incubated with chromatin at 4 °C with rotation overnight. Chromatin was washed, crosslink was reversed at 65 °C overnight and DNA was isolated using Agencourt AMPure magnetic beads (Beckman Coulter). Subsequently, qPCR was performed (StepOne, Applied Biosystems) using ChIP and input DNA amplifying different regions around the transcriptional start site of Acod1 (Irg1). Enrichment of TFEB binding was calculated as ChIP–DNA relative to input-DNA PCR signal for each primer pair and normalized to a negative control region (non-accessible heterochromatin region).