GSM3825629: TKO ESC 5hmC-RIP Input repl 1; Mus musculus; RIP-Seq
Total RNA from WT ESCs, TKO ESCs and WT EBs were extracted with either the RNeasy Mini or Maxi kit or TRIzol according to the manufacturer's instructions. Genomic DNA was eliminated by DNase treatment. hMeRIP-seq: The procedure was performed in ESCs as described in Dominissi et al., 2012. Briefly, 1 mg of total RNA was sonicated to an average size of 200-300bp. RNA was then precipitated in ethanol, resuspended in RNase-free ddH2O and the fragmentation efficiency was checked on a Bioanalyzer RNA chip. For immunoprecipitation, RNA fragments were denatured by heating at 70°C for 5 min, chilled on ice for 5 min, then incubated overnight at 4°C with or without 12.5 ug of anti-5hmC antibody, in freshly prepared 1X IP buffer (50 mM Tris-HCl pH=7.4, 750 mM NaCl and 0.5% Igepal CA-630, RNasin 400 U/ml and RVC 2 mM) supplemented with protease inhibitors. Samples were then incubated at 4°C for 2.5h with 60 ul equilibrated Dynabeads Protein G, washed three times for 5 min with 1 ml IP buffer, and eluted by addition of 1 ml TriPure Reagent followed by RNA extraction according to the manufacturer's instructions. Library preparation was performed as described in Delatte et al., 2016. Briefly, 5 to 10 ng dsDNA was subjected to 5' and 3' protruding end repair, followed by the addition of non-templated adenines to the 3' ends of the blunted DNA fragments, which allows ligation of Illumina multiplex adapters. The DNA fragments were then size selected so as to remove all unligated adapters and to sequence 200-300-bp fragments. 18 cycles of PCR were carried out to amplify the library. DNA was quantified by fluorometry with the Qubit 2.0 and DNA integrity was assessed with a 2100 bioanalyzer. 6 pM of DNA library spiked with 1% PhiX viral DNA was clustered on cBot and then sequenced.