Our team has implemented several state-of-the-art techniques that are essentials for completing our current project successfully.
Ribosome profiling or Ribo-sequencing (Ribo-seq). This technique is based on deep sequencing of ribosome-protected mRNA fragments and permits genome wide analysis of translated sequences at the sub-codon resolution.
Ribo-seq procedure. A. (step 1) Cells are treated with translation inhibitor. (step 2) Cells are lyzed and mRNA-ribosome complexes are isolated using sucrose gradient density centrifugation (Fig.4A). (step 3) RNase I digests the RNA not protected by ribosomes. (step 4) The RNA fragments (ribosome profiling fragments, RPF) are size-selected, ligated, and reverse transcribed to make cDNA libraries for sequencing. Sequence results alignment to genomic sequence permits to establish translational profile. B. Gel migration on TBE urea-gel of RNase treated sample (step 3). RPF (about 28nt) is shown in the red square. C. Analysis of RPFs obtained from recent Ribo-seq experiment from Team 1. All reads are mapped on mRNA. The x-axis represents the distance between the 5′ extremity of RPF and the A of the AUG of initiation. Each dot is colored depending on its phase. The read density at each nucleotide position is the sum of mapped reads. This shows clear difference between before and after “start of translation” (with one main frame in blue) and validates Ribo-seq procedure.