GSM6765081: RIBO_pH4.4_1; Escherichia coli str. K-12 substr. MG1655; OTHER
Cells were flash frozen in liquid nitrogen and lysed by cryogenic grinding in a SPEX SamplePrep 6875 Freezer/Mill. Polysome fractions were isolated using ultracentrifugation. Nuclease digestion was performed using MNase (NEB). Monosomes were recovered using MicroSpin S-400 HR columns (GE Healthcare) and RNA was isolated using the miRNeasy Mini Kit (QIAGEN) in combination with the RNase-Free DNase Set (QIAGEN). The isolated RNA was loaded on a 15% TBE Urea size selection gel and after staining with SYBR Gold (Invitrogen), ribosomal footprints between 15 and 45 nt were excised from the gel. Gel pieces were crushed and RNA was recovered by precipitation using Corning-Costar Spin-X Centrifuge Tube Filters. Isolation of RNA fragments corresponding to ribosomal footprints of 15 – 45 nt size was verified using the RNA 6000 Nano Kit (Agilent). 5´ phosphorylation of RNA fragments was achieved using T4 Polynucleotide Kinase (NEB) and Adenosine 5´-Triphosphate (ATP) (NEB). After RNA recovery using an RNA MinElute Cleanup Kit (QIAGEN), footprint fragments were again evaluated using the Agilent 2100 Bioanalyzer. cDNA libraries were constructed using the NEBNext Small RNA Library Prep Set for Illumina (NEB) with modifications described in Latif et al. 2017 DOI: 10.2144/000114302. cDNA library quality was assessed by using a High Sensitivity DNA Kit (Agilent).