GSM2067512: unc-75 CLIPseq; Caenorhabditis elegans; OTHER
Worms were lysed by sonication in Homogenization Buffer (100 mM NaCl, 25 mM HEPES, 250 μM EDTA, 2 mM DTT, 0.1% NP-40, 25 units/ml RNasin and Protease Inhibitors). Lysates were centrifuged at 16,000g for 15 min at 4°C and supernatants collected and incubated with M2 magnetic beads (Sigma) overnight on a rotator. Samples were treated with micrococcal nuclease followed by phosphatase treatment, linker addition and 5’-labeling with 32P-γ-ATP. Complexes were eluted and separated with gel electrophoresis. A band corresponding to UNC-75/RNA complexes was cut out of the membrane and proteins degraded by Proteinase K. Associated RNAs were reverse-transcribed and amplified by PCR for deep sequencing.
GEO Accession
GSM2067512