GSM5737990: RPF_CFBE_R553X-CFTR_supp tRNA; Homo sapiens; OTHER
The whole organs were pulverized in liquid nitrogen and lysed by grinding in 360 µL 10 mM Tris-HCl (pH 7.4) supplemented with 5 mM MgCl2, 100 mM KCL, 1% NP-40, 2mM DTT, 100 µg/mL cycloheximide topped with 40µL of 10% sodium deoxycholate. Lysate from each animal organ were used to produce an independent library. re flash frozen and immediately lysed by grinding in 10mM Tris-HCL pH 7.4, 5mM MgCl2, 100mM KCL, 1% NP-40, 2% sodium deoxycholate.The lysates from three animals each were pooled. Isolation of mRNA-bound ribosome complexes, RNase I digestion-derived ribosome-protected fragments (RPFs), RNA-size selection and generation of the cDNA libraries was performed as described (Guo et al, 2010; doi: 10.1038/nature09267 )