GSM8190831: AAV MCHERRY-FTO_2_input; Rattus norvegicus; RIP-Seq
This analysis was performed at the Sheba medical center on RNA extracted from rat primary neuronal cultures (2 x 10^6 cells) using Pure Link™ RNA Mini Kit (Invitrogen™), with on-column DNase treatment (according to the kit's protocol) and assessing the integrity of the RNA using a Agilent Bioanalyzer 2100z. The mRNA was enriched (300-500 ng on average) from the total cell RNA using the Gene Elute mRNA miniprep kit (#MRN70, Sigma-Aldrich) and it was then fragmented to ~80 nts on average using RNA Fragmentation Reagents (Ambion, # AM8740). The fragmented RNA was subjected to m6A precipitation using Protein G Dynabeads (cat.# 10003D, Life Technologies) coupled to a m6A antibody (Synaptic Systems, #202003). Immunoprecipitation (IP) was performed over 3 hours on a rotator at 4 ºC in freshly prepared IP buffer composed of 10 mM Tris [pH 7.4], 150 mM NaCl and 0.1% Igepal CA630, and containing the RNasin Plus RNase Inhibitor (# N2615, Promega). Prior to immunoprecipitation, the RNA was denatured for 2 minutes at 72 ºC and subsequently, the m6A RNA was eluted using Proteinase K (100 µg: cat. # P4850, Sigma, new 1.03.2017, 4ºC) in a buffer containing 5 mM Tris [pH 7.5], 1 mM EDTA [pH 8] and 0.25% SDS. The RNA samples were incubated for 90 minutes at 37 ºC with vigorous rotation (900 rpm) using a thermomixer and the supernatant containing the eluted m6A RNA fragments was transferred to a new tube containing 1 ml Trizol. The aqueous phase was then loaded onto the Zymo RNA Clean & Concentrator™ kit. Libraries were constructed using NEB Next Multiplex Oligos for Illumina, and a NEB Next® and Ultra II Directional RNA Library Prep Kit for Illumina® following the manufacturer's instructions. The cDNA libraries synthesized were resolved on Tape station (Agilent), demonstrating an average size of ~300 bps. Multiple libraries with different barcodes (at 3′ end) were mixed in equimolar ratios and sequenced with the NovaSeq 6000 Illumina Platform (Sheba Cancer Research Center, Sheba Medical Center, Israel) at length of 150 paired end (75 nts in each). The library strategy involves mRNA enrichment followed by m6A immunoprecipitation to isolate RNA fragments containing N6-methyladenosine modifications. These fragments are then used for library construction, where cDNA libraries are prepared and sequenced using Illumina technology. The strategy aims to capture m6A-modified RNA transcripts for downstream analysis, providing insights into epitranscriptomic regulation in rat primary neuronal cultures.