title: GSM4478905: m6A-RIP-Seq N2 embryos; Caenorhabditis elegans; RIP-Seq
library_construction_protocol: mRNA was obtained from total RNA (100 ug) by purifying twice with a rRNA depletion kit (RiboMinus Eukaryote Kit v2) following manufacturer's instructions.   m6A RIP-seq experiments were performed as previously described (Sendinc, David-Valle et.al, Molecular Cell, 2019). Briefly: 5 g of mRNA are fragmented at 98ºC for 3 min in 200 ul of fragmenting buffer (10 mM Tris pH 7.4, 10 mM ZnCl2) and quenched with 70mM EDTA. 50 L of Protein G Dynabeads (Invitrogen) are incubated with 250 ug of rabbit monoclonal anti-m6A (Cell Signaling Technologies, Cat #56593S) at 4ºC for 30 mins. The beads/antibody slurry is added to the fragmented mRNA and incubated in rotation for 1 h at 4ºC. Beads were washed twice with 200 L of reaction buffer (150 mM NaCl, 10 mM Tris-HCl, 0.1% Triton X-100, pH 7.5), twice with 200 L of low salt buffer (50 mM NaCl, 10 mM Tris-HCl, 0.1% Triton X-100, pH 7.5) and twice with 200 L of high salt buffer (500 mM NaCl, 10 mM Tris-HCl, 0.1% Triton X-100, pH 7.5). Immunoprecipitated RNA was eluted in TRIzol and purified with the RNA clean and concentrator Kit -5 (Zymo, Cat #R1013). Illumina sequencing libraries are prepared with the NEBNext Ultra II Directional Library Prep Kit for Illumina from NEB (#E7760), following manufacturer's instructions. RNA-Seq libraries are generated from three biological replicates for Input (RNA-Seq) and m6A-immunoprecipitated mRNA. Libraries are sequenced in a NextSeq 550 with an average coverage of 30 million reads per sample.
attributes.tag: GEO Accession
attributes.value: GSM4478905