GSM2500126: Vibrio_natriegens_ribosome_profiling_MOPS_comp_3NaCl; Vibrio natriegens NBRC 15636 = ATCC 14048 = DSM 759; OTHER
Extraction was performed as described in detail previously (Li et al., Cell 2014). 100 mL of cell cultures was rapidly filtered at 37C by passing through a nitrocellulose filter. Cell pellets was were rapidly collected using a pre-warmed metal table crumber, flash frozen in liquid nitrogen, and combined with frozen droplets of lysis buffer. Cells and lysis buffer were pulverized in 10 mL canisters (Retsch) pre-chilled in liquid nitrogen using Qiagen TissueLyser II. Pulverized lysate was thawed on ice and clarified by centrifugation at 4C. Ribosome protected mRNA fragments were size selected via gel purification and dephosphorylated at the 3' end. cDNA library preparation then proceeded using the SMARTer smRNA-seq kit from Clontech following the manufacturer's instructions.