GSM2055261: DMS-seq 37°C in vitro WT; Escherichia coli; OTHER
Extraction was performed as described in detail previously (Li et al., 2012; Oh et al., 2011). 200 ml of cell culture was rapidly filtered at 37°C 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. 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,. Cell 147, 1295 (2011)]. More at G.W. Li, D. Burkhardt, C.A. Gross, J. S. Weissman, Cell 157, 624-635 (2014).
GEO Accession
GSM2055261