GSM9035396: HG001, Total RNA-seq associated to RNase 1 Ribo-seq, no drug, rep1 sample; Staphylococcus aureus; RNA-Seq
Cultures were subjected to rapid cooling by swirling in an ice-bath for 3 min, before 40 mL of each sample (split into two 50 mL falcon tubes) were harvested by centrifugation. Another 10 mL of each culture were flash frozen in liquid nitrogen and reserved for total RNA sequencing. For Ribo-seq and Ribo-Ret, cell pellets were resuspended in 550 μL of pre-chilled lysis buffer A (20 mM Tris-HCl pH 8, 50 mM MgCl2, 100 mM NH4Cl, 5 mM CaCl2, 0.4 % Triton X-100, 0.1 % NP-40, 100 U/mL DNase 1, 1 mM GMPPNP). Mechanical lysis was performed by two rounds of 'fastprep' bead-beating at 6 m/s for 40 seconds in 2 mL fastprep tubes containing Lysing Matrix B. Subsequently, samples were centrifuged at 16,000 g for 5 min at 4°C and supernatants were carefully retrieved. Measurement of absorbance at 260 nm in a Nanodrop spectrophotometer served to adjust nuclease concentration for subsequent digestion by either MNase or RNase 1. For MNase digestion, 750 U of MNase and 50 U of SUPERase-In (does not inhibit MNase) were added per 40 absorbance units (AU). After 1 h shaking at 1400 rpm and 25°C, the reactions were quenched by addition of 5 mM EGTA (f.c.). For the alternative digestion by RNase 1, 250 U of RNase 1 were added per 40 AU of sample concentration. The digestion was performed for 20 min shaking at 800 rpm and 37°C, before it was stopped by addition of 100 U of SUPERase-In. All samples were kept strictly on ice prior to loading onto a 5-50 % sucrose gradient for isolation of 70S monosome peaks. Ribosome protected fragments (RPFs) were extracted using acidic hot phenol and size selected on a 15% polyacrylamide- 8 M Urea gel (PAGE). RPFs were gel eluted via passive elution in RNA elution buffer (0.5 M NH4Ac pH 6.5, 1 mM EDTA, 0.1 % SDS) o/n at 4°C shaking at 700 rpm prior to acidic PCI (phenol/chloroform/isoamyl alcohol, 25/24/1) extraction and ethanol RNA precipitation. All samples were subjected to ribosomal RNA depletion employing a commercially available rRNA depletion kit (siTOOLs) according to the manufacturer's protocol. Associated total RNA sequencing libraries were then prepared using the NEBNext Ultra II Directional RNA Library Prep Kit following the manufacturer's instructions.