GSM3656945: CML-B input; Mus musculus; RIP-Seq
blast crisis CML cells were freshly harvested, crushed in IMDM + 3% FBS, passed through 40 uM cell strainers followed by red blood cell lysis. Cells were incubated with a cocktail of lineage specific antibodies (CD5, CD11b, CD19, B220, Gr-1 and TER119, Stemcell Technologies) for negative selection of Lin- cells using an EasyStep immunomagnetic column (Stemcell Technologies). 20 million cells were per sample were subsequently washed in PBS and UV-crosslinked at 400mJ/cm2 on ice. Samples were then pelleted, snap-frozen and stored at -80°C. Pellets were lysed in iCLIP lysis buffer, treated with RNAse I for 5 min at 37°C, followed by immunoprecipitation using 10 μg anti-ELAVL1 mouse monoclonal IgG (ab170193, Abcam) and 125 μL M-280 Sheep-α-Mouse IgG Dynabeads (ThermoFisher #11201D) per sample. After stringent rounds of washing, samples were dephosphorylated (FastAP; ThermoFisher, T4 PNK; NEB), 3' ligation (on-bead) with a barcoded RNA adapter followed using T4 RNA Ligase (NEB). Samples were again stringently washed, run on standard protein gels and transferred to nitrocellulose membranes. The region spanning 36-115 kDa was then isolated, followed by extraction of RNA from the membranes and reverse transcription (AffinityScript; Agilent). A DNA adapter containing a 5' random-mer was then ligated (3'), followed by cleanup of the samples and PCR amplification. Libraries were size-selected (175-350 bp) and purified from 3% low melting temperature agarose gels, followed by sequencing on the Illumine HiSeq 2500 platform (paired-end).
GEO Accession
GSM3656945