GSM8340258: SUM1315, Normoxia Polysomes; Homo sapiens; RNA-Seq
Cells were exposed to hypoxic or normoxic conditions and then treated with cycloheximide before collection. After washing with cold PBS containing cycloheximide, the cells were lysed using a polysome lysis buffer. The lysates were incubated at 4°C and centrifuged to determine protein concentrations. Equal amounts of protein were applied to sucrose density gradients and centrifuged. The gradients were analyzed using the Brandel BR-188 system. RNA was isolated from the polysomes using the RNeasy Mini Kit, and 500 ng of total RNA was used for library construction and sequencing. RNA library preparations and sequencing were performed using the NEBNext Ultra RNA Library Prep Kit. Libraries were validated on the Agilent TapeStation and quantified with the Qubit 2.0 Fluorometer and qPCR.