GSM3825611: Tet2-KI ESC Tet2-RIP Input repl4; Mus musculus; RIP-Seq
Total RNA from WT ESCs 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. 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 on a HiScanSQ module.