GSM2801049: DMS-seq of purified mRNA in vitro at 10°C, rep2; Escherichia coli str. K-12 substr. MG1655; OTHER
Extraction was performed as described in detail previously (Li et al., 2012; Oh et al., 2011; Rouskin et al., 2014). For ribosome profiling, 200 ml of cell culture was rapidly filtered 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 4°C. Lysate containing 0.5 mg of RNA was digested for 1 h with 750 U of micrococcal nuclease (Roche) at 25°C. The ribosome-protected RNA fragments were isolated using a sucrose gradient followed by hot acid phenol extraction. mRNA fragments were size selected via gel purification, and ligated to 5' adenylated DNA oligo. After reverse transcription, the single stranded DNA was circularized, and PCR amplified (Oh et al., 2011; Li et al., 2014; Rouskin et al., 2014).
GEO
GSM2801049