|Number of samples:||8|
|Release date:||Nov 27 2018|
|Last update date:||Nov 29 2018|
|Dataset link||Divergent rRNAs as regulators of gene expression at the ribosome level|
Ribosome profiling analysis. Cells were grown at 30 °C in LBS media containing 0.2 μg/ml tetracycline (Tc) to an OD600 of 0.7. After treatment with 100 µg/ml chloramphenicol to stop translation, the cells were incubated at 30 °C for 5 min. The culture was cooled on ice and harvested by centrifugation. The pellet was washed with pre-chilled buffer A (50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 100 mM NH4Cl, 6 mM 2-mercaptoethanol, 0.5 mM EDTA) containing 1 mM of chloramphenicol and was lysed using a French press. Cellular debris was removed by centrifuging for 30 min at 30,000 × g. The clarified lysate (supplemented with 5 mM CaCl2) was digested with micrococcal nuclease for 1 h at 25 °C and quenched with EGTA to a final concentration of 6 mM. The resulting lysate was loaded onto a 30% sucrose cushion. Crude ribosomes were pelleted by centrifugation at 100,000 × g for 16 h at 4 °C. The pellets were resuspended and loaded onto a 15–40% sucrose gradient and centrifuged at 79,500 × g for 13.5 h at 4 °C. After centrifugation, monosomes were obtained by fractionation and measurement of optical density at 260 nm. Monosomal RNA was obtained by phenol extraction and ethanol precipitation and was resuspended in RNase-free water. To obtain ribosome-protected mRNAs, extracted monosomal RNA was resolved on a 15% polyacrylamide-8 M urea-TBE gel. A band between 28 and 42 bp was excised, gel puriﬁed, precipitated, and resuspended in RNase-free water. For ribosomal RNA depletion the Ribo-Zero rRNA removal kit was used according to the manufacturer’s instructions. Isolated RNAs were used to construct a cDNA library for Illumina sequencing using the TruSeq Small RNA Sample Prep Kit according to the manufacturer’s instructions. RNA sequencing was performed on the Illumina HiSeq 2500 platform using single-end 50 bp sequencing. Transcriptome analysis. Total cellular RNA was extracted from cells grown at 30 °C in LBS media containing 0.2 μg/ml tetracycline (Tc) to an OD600 of 0.7 using TRIzol according to the manufacturer’s instructions. The mRNA in total RNA was converted into a library of template molecules suitable for subsequent cluster generation using the reagents provided in the Illumina TruSeq RNA Sample Preparation Kit. The first step in the workflow involves removing the rRNA in the total RNA using Ribo-Zero rRNA Removal kit. Following this step, the remaining mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library. RNA sequencing was performed on the Illumina HiSeq 2500 platform using paired-end 100 bp sequencing. Sequencing data analysis. The sequence of the reference genome was retrieved from the NCBI database. RNA-seq reads were aligned to the reference genome sequence using Bowtie2 or BWA. Coordinates of mapped reads were obtained by processing BAM formatted files using BEDOPS tools and were stored in BED formatted files. Coordinates of genome features such as protein coding genes were extracted from the genome annotation data. Boundaries of features such as transcription start sites and gene segments were prepared: a transcription start site was defined as positions from -60 to +60 (the first nucleotide of a start codon was numbered as +1) and a gene segment as positions from -60 to the end of the stop codon. Overlapping genome-mapped RNA-seq reads were assigned to these features. The normalized abundance of reads was measured as number of reads per million mapped reads (RPM).