title: GSM5833456: Ythdc-RNAi basal RNA-seq rep3; Drosophila melanogaster; RNA-Seq
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). 1.4ul of NEB m6A antibody (NEB, E1610S), or 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). Libraries were made using SMARTer Stranded Total RNA-Seq Kit V2 without rRNA depletion (takarabio, 634411) for IPed and input RNA, and sequenced using illumina HiSeq with 40M paired end reads (2x150bp). Library preparation and sequencing was done by Admera Health. Three biological replicates per genotype and condition were done with NEB m6A antibody, and two biological replicates were done with Synaptic Systems m6A antibody. 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). Three biological replicates were done for each experimental timepoint, condition, and genotype. Technical repeats and repeat additional biological replicates were done as indicated. Libraries were made using SMARTer Stranded Total RNA-Seq Kit V2 without rRNA depletion (takarabio, 634411) for IPed and input RNA, The RNA-seq libraries from brains were prepared using the Tru-seq stranded mRNA library prep. Both m6A-IP and RNa-seq were sequenced using illumina HiSeq with 40M paired end reads (2x150bp).