title: GSM2679696: Hfq mut exp mRNA; Escherichia coli; RNA-Seq
library_construction_protocol: Total RNA was extracted using TRI Reagent (Sigma Aldrich), enriched by depleting small RNAs with GeneJET Purification Kit (Fermentas) and rRNA with MICROBExpres Bacterial mRNA Enrichment Kit (Ambion) and fragmented in alkaline solution (2 mM EDTA and 100 mM Na2CO3 pH 9.2 for 40 min at 95°C) to fragments with size of 24-35 nts. For RPFS, cells were lysed by freeze-rupturing (Retch Mill) and 100 A260 units of ribosome-bound mRNA fraction were directly used for polysomal analysis or subjected to nucleolytic digestion with 10 units/µl micrococcal nuclease (Fermentas) for 10 min at room temperature in buffer with pH 9.2 (10 mM Tris pH 11 containing 50 mM NH4Cl, 10 mM MgCl2, 0.2% triton X-100, 100 µg/ml chloramphenicol and 20 mM CaCl2) to obtain the monosomal fraction. Separation was obtained by sucrose density gradient (15-50% w/v). Subsequently, 20-35-nt RNA fragments from the monosomal fraction were size selected on a denaturing 15% polyacrylamide gel. RNA-size selection and generation of the cDNA libraries was performed as described {Ingolia, 2009}
attributes.tag: GEO Accession
attributes.value: GSM2679696