GSM4421260: YTHDF_NP3_Df_1Whead_rep2; Drosophila melanogaster; RNA-Seq
Briefly, total RNA was collected from <1 week old w1118 (wild type) and mettl3[null] (mutant) female heads using TRIzol RNA extraction. miCLIP: Poly(A)+ RNA was enriched using two rounds of selection.RNAs were fragmented, incubated with the α-m6A (202 003 Synaptic Systems) and crosslinked twice in a Stratalinker 2400 (Stratagene) using 150 mJ per cm2. Crosslinked RNAs were immunoprecipitated using Protein A/G magnetic beads (Thermo) and washed under high salt conditions to reduce non-specific binding. Samples were radiolabeled with T4 PNK (NEB), ligated to a 3' adaptor using T4 RNA Ligase I (NEB), and purified using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and nitrocellulose membrane transfer. RNA fragments containing crosslinked antibody peptides were recovered from the membrane using proteinase K (Invitrogen) digestion. Recovered fragments were subjected to library preparation. First-strand cDNA synthesis was performed using SuperScript III (Life Technologies) and iCLIP-barcoded primers, which contain complementarity to the 3' adaptor on the RNA. cDNAs were purified using denaturing PAGE purification, circularized using CircLigase II (EpiCentre), annealed to the iCLIP Cut Oligo, and digested using BamHI (Thermo). To generate libraries for sequencing, the resulting linear cDNAs were amplified using Accuprime SuperMix I (Invitrogen) and P5 and P3 Solexa primers, and purified using Agencourt AMPure XP beads (Beckman Coulter). For input libraries, poly(A)+ RNAs were fragmented and directly subjected to radiolabelling and 3' adaptor ligation. All subsequent steps are as listed above. Libraries were paired-end sequenced on an Illumina HiSeq2500 instrument at the New York Genome Center (NYGC). RNA-seq: Sequencing libraries were prepared using the TruSeq Stranded Total RNA Sequencing Kit (Illumina) following the manufacturer's protocol miCLIP-seq and RNA-seq
GEO Accession
GSM4421260