GSM3611772: mES ΔSX input; Mus musculus; RIP-Seq
Standard Trizol extraction Total mES RNA was subjected to one round of Poly(A)Purist MAG treatment to enrich for polyadenylated RNAs as per manufacturer's instructions (Ambion). RNA was then fragmented to 100nt median sized fragments using RNA Fragmentation Reagents (Ambion) and subjected to one round of m6A immunoprecipitation. For immunoprecipitation of RNA, 5 ug of m6A antibody (Millipore) was coupled to 40 ul Protein A Dynabeads (Novex) in 100 ul 1X IPP Buffer (50mM Tris-HCl, pH 7.5; 150mM NaCl; 0.1% NP-40; 5mM EDTA) overnight at 4C. Beads were then washed twice in 1X IPP Buffer. Fragmented RNA was denatured at 70C for 2 min, cooled on ice, and bound to antibody-beads in 185 ul 1X IPP Buffer for 3h at 4C. Beads were then washed sequentially with (2x) 500 ul 1X IPP Buffer, (2x) 500 ul Low Salt Buffer (0.25X SSPE; 1mM EDTA; 0.05% Tween-20; 37.5mM NaCl), (2x) 500 ul High Salt Buffer (0.25X SSPE; 1mM EDTA; 0.05% Tween-20; 137.5mM NaCl), (1x) 500 ul TET Buffer (10mM Tris-HCl, pH 8.0; 1mM EDTA; 0.05% Tween-20). Beads were eluted with 50 ul RLT Buffer (Qiagen RNeasy Mini Kit) and incubated at 25C for 5 min, and recovered with RNeasy Mini Kit followed by concentrating with Zymo RNA Clean & Concentrator in 10 ul water. 10 ng of input RNA (before immunoprecipitation) and 10 ng of immunoprecipitated RNA were then used to prepare sequencing libraries using SMARTer Stranded RNA-Seq Kit - Pico Input Mammalian (Clontech #634411), as per manufacturer's instructions. Sequencing libraries were pooled and sequenced on the Illumina MiSeq (files named *apr26*, including the following samples: ) and NextSeq (files named *jun24*, including the following samples).
GEO Accession
GSM3611772