Ultrapure water

The SYBR Green RT-qPCR assay (one step) was performed, according to the manufacturer’s recommendations, with the QuantiFast SYBR® Green RT-PCR kit (QIAGEN, Hilden, Germany). Briefly, 8 μL of target RNA and 1 μL [10pM/μL] from each primer (E-Sarbeco F1 and R2) [5 (link)] were added to the 12.5 μL 2x Master Mix QuantiFast SYBR Green RT-PCR, 0.25 μL of enzyme RT-Mix and Ultra-Pure Water (Thermo Fisher Scientific, Waltham, MA, USA) to complete 25 μL of reaction. The reverse transcription reaction was performed at 50 °C for 10 min, followed by 5-min denaturation at 95 °C, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. The reaction was completed by determining the dissociation curve of all amplicons generated in the ABI 7500 device (Applied Biosystems, Weiterstadt, Germany). The SYBR Green qPCR reactions (two steps) were carried out in a 20-μL final volume containing 10 μL of 2X PowerUp SYBR Green PCR Master Mix (Applied Biosystems, Weiterstadt, Germany), 2 μL of each primer [10 pM/μL], 5 μL of cDNA, and 1 μL of Ultra-Pure Water (Thermo Fisher Scientific, Waltham, MA, USA). The thermal cycling conditions used were as follows: 50 °C for 2 min, followed by 5 min denaturation at 95 °C, followed by 40 cycles at 95 °C for 15 s, 58° for 15 s, and 72 °C for 1 min, and a dissociation curve was subsequently made. Both reactions were performed using an ABI 7500 machine (Applied Biosystems).

1 μg of genomic DNA was diluted to 130 μl ultrapure water (Invitrogen) and sheared with an average size about 300bp prior to the capture. m6dA captured was performed using a m6dA antibody (Active Motif) to capture m6dA enriched genomic regions. The procedure was adapted from manufactory’s protocol for Methyl DNA immunoprecipitation (Active motif). 500 ng of sheared DNA and 4 μg of m6dA antibody was used for each immunoprecipitation reaction and all selected targets (GATC site proximal BDNF P4: Chr2: 109692436–109692774; distal GATC site: Chr2: 109691953–109692103) were normalized to input DNA and then to their own controls by using the _ΔΔCT method, and each qPCR reaction was run in duplicate for each sample and repeated at least twice. Samples that didn’t reach data from IgG enrichment is excluded.

All chemicals were obtained from Sigma-Aldrich unless otherwise stated. 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt)(DPPE-PEG₂₀₀₀) was purchased from Avanti Polar Lipids. fSP (Poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}]) was purchased from Luminescence Technology Corp. 2-Methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA) was purchased from TCI America. JC-1 dye was purchased from Invitrogen. ATN224 was purchased from Cayman Chemical Company. XenoLight™ D-Luciferin was purchased from Perkin Elmer. TRIzol® reagent was purchase from Thermo Fisher Scientific. Ultrapure water was purchased from Invitrogen. 1× PBS, 1× D-PBS, and 1× HBSS were purchased from Gibco. Sterile 0.9 % saline solution was purchased from TEKnova. CCL-1 was synthesized with the procedures reported previously.^{58 (link)}