library_construction_protocol: For m6A-IP seq flies were flash frozen and head tissue was collected into cold RNAse free tubes. Total RNA was extracted from 200 Drosophila Heads per replicate using Trizol/ chloroform extraction. PolyA+ mRNA was obtained using NEBNext Poly(A) mRNA Magnetic Isolation Module. PolyA+ RNA was fragmented using the NEB Next Magnesium Fragmentation Module (NEB, E6150S) for 4 minutes at 95 °C for a 250 ng sample of polyA+ RNA, and RNA was repurified using the Zymo RNA clean & concentrator -5 kit (Zymo, R1013). 10% of the fragmented polyA+ RNA was saved as an input control for sequencing. M6A -immunoprecipitation was done using the EpiMark N6-Methyladenosine Enrichment kit protocol with some minor alteration. 30ul of protein G magnetic beads (NEB, #S1430) were washed and resuspended in IP buffer (150mM NaCl, 10mM Tris-HCL, 0.1% NP-40). 4ul of synaptic systems antibody (SYS, 202003) was conjugated to protein G-magnetic beads (NEB, S1430S) for 2 hours at 4°C. Beads/ antibody were washed twice in IP buffer. ~1μg PolyA+ RNA was incubated with beads/antibody in IP buffer supplemented with 0.1% SUPERase-In RNase Inhibitor (Thermo Fisher; AM2696) for 2 hours at 4°C. After incubation, RNA/beads/antibody are washed twice in IP buffer, twice in low salt IP buffer (50mM NaCl, 10mM Tris-HCL, 0.1% NP-40), and twice in high salt IP buffer (500mM NaCl, 10mM Tris-HCL, 0.1% NP-40). RNA is eluted from beads with 25μl of RLT buffer twice and elution was pooled and concentrated using Zymo RNA clean and concentrator kit-5 (R1015). Library preparation and sequencing was done by Admera Health. Total RNA was extracted from brains using trizol/chloroform and Zymo RNA clean and concentrator kit-5 (R1015). The RNA-seq libraries from brains were prepared using the Tru-seq stranded mRNA library prep. Library preparation and sequencing was done by Admera Health, and sequenced using illumina HiSeq with 40M paired end reads (2x150bp). Biological replicates were done for each experimental condition and genotype as indicated. For FACS sorting Brains (10–15 per replicate) from young (5days old) and old (34days old) males were dissected, collected into a tube with cold Schneider's medium (SM) containing 45μM actinomycin D, and washed twice in PBS. Next, we added 300μL of dissociating solution, which included Papain diluted in PBS (Worthington PAP2, LK003178) and liberase (2.5 mg/ml). Brains were dissociated at 25°C using a shaker at 1000rpm for 20 min and vigorous pipetting every 5min, and passing through 25G 5/8 needle 7x. Dissociated tissue was filtered through 40μm cell strainers and sorted on an BD FACS Aria II SORP with a 100μm nozzle into a 96 well plate at the sorting facility of the University of Pennsylvania. Dead cells were excluded by staining with 4,6-diamidino-2-phenylindole (DAPI). GFP+ cells gates were set according to the fluorescence profile of GFP- brain tissue, and nuclear stain Syto60. Desired glia or neuronal populations (~500 cells per replicate) were directly sorted into 10X lysis buffer (Takara 635013) plus RNAse inhibitor for library preparations using SMART-seq v4. For TRAP IP fly brains (30 brains/sample) were dissected and stored in buffer ( 10mM Hepes, 150 mM KCl, 5mM MgCl2, 100ug/ml Cyclohexamide, 1x protease inhibitor, Rnase inhibitor) in -80 until samples were ready to process. Protein G Magnetic beads were coated with 4 ug of anti-flag antibody, and resuspended in reaction buffer (10mM Hepes, 150 mM KCl, 5mM MgCl2, 100ug/ml Cyclohex, 1x protease inhibitor (Sigma, 11873580001), + 0.1% triton x-100, Rnase inhibitor (Thermo Fisher, AM2696). Samples with brain tissue were homogenized in lysis buffer (reaction buffer + 0.5% triton-x). 20% of sample was saved as input control and RNA extraction using zymo clean and concentrator kit -5. The rest of the sample was added to antibody plus beads, and incubated for 4 hr at 4°C. Beads + RNA was washed and RNA was extracted from beads using zymo clean and concentrator kit -5. Libraries were made using SMARTer Stranded Total RNA-Seq Kit V4 for IPed and input RNA, and sequenced with 40M paired end reads (2x150bp). For experiment 21 and 08, m6A-IPseq libraries were made using SMARTer Stranded Total RNA-Seq Kit V3 without rRNA depletion (takarabio, 634486) for IPed and input RNA. Experiment 07 brain RNA-seq libraries were prepared using the Tru-seq stranded mRNA library prep. Experiment 25, 27, and 29 FACS sorting and TRAP-seq libraries were made using SMART-Seq V4 + NexteraXT Library Preparation. All libraries were sequenced using illumina Illumina NovaSeq 6000 with 40M paired end reads (2x150bp).