Pncrs_CLIP_HPNE_01Feb2013
hTERT-HPNE and MIA PaCa-2 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and 1 % penicillin/streptomycin. Cells were washed twice with phosphate buffered saline (PBS) and UV irradiated at 254nm for a total energy deposition of 600 mJ/cm2. Brain tissues were placed into Hank's Balanced Salt Solution (HBSS) complete with protease and RNAse inhibitors and triturated through 18, 20, and 22 gauge needles prior to UV irradiation at 254nm for a total energy deposition of 1200 mJ/cm2. Cells and tissues were lysed in PXL buffer and Ago-RNA complexes were immunoprecipitated using anti-pan-Ago antibody 2A8 as per Chi et al. Immunoprecipitated RNAs were digested using RNAse T1 (100U/μl) and isolated from protein G Dynabeads (Life Technologies) by phenol-chloroform extraction. Sequencing libraries were constructed from extracted RNAs using the SOLiD Small-RNA Expression Kit (Life Technologies) as per manufacturer instructions. Unpaired reads were sequenced using the Applied Biosystems 5500xl Genetic Analyzer. Colorspace adapter sequence is (330201030313112312).