GSM7567432: Ecoli LB06 Capture Rep2; Escherichia coli BW25113; ncRNA-Seq
RNA isolation with Trizol reagent, precipitation with GlycoBlue Sequencing libraries were prepared from inputs and captured s4u-containing RNA. 3' end adaptors (Table 6) were ligated 100 ng of RNA in an overnight reaction at 16C (250 nM adaptor, 1x T4 RNA ligase buffer, 1 ml of T4 RNA Ligase 1 (ssRNA Ligase), High Concentration (M0437M), 50 mM DTT (Sigma 43816-10mL), 6 ml of 50% PEG 8000 (NEB B1004A), 40 units of Ribolock RNase inhibitor EO0382). All samples were then gel purified on a denaturing 10% polyacrylamide gel (National Diagnostics, EC-829). Gels were cut between 60 to 200 nt and purified via crush and soak overnight tumble at 4C (crush gel through needle punched bottom of 500 ml tubes into 1.7 mL Eppendorf tubes, soak in 10 mM Tris pH 7.5 (15567-027), 500 mM NaCl (AM9759), 1 mM EDTA (AM9261), 0.1% SDS (351-032-101). Gel buffer mix is then transferred to SpinX columns (CLS8162-96EA) to remove remaining gel. Three volumes of 100% ethanol are added to the RNA-containing buffer for precipitation.