RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique, also called RIBO-seq, is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method is its ability to monitor translation by precisely mapping the position and number of ribosomes. RIBO-seq is based on the fact that the ribosome, by binding to the mRNA molecule, protects the buried fragment of the transcript upon ribonuclease digestion.

The obtained fragments, called ribosomal footprints, can be sequenced and mapped onto the transcript they originated from, resulting in determination of the exact position of the ribosomes. Simultaneously to the RIBO-seq, high throughput total mRNA sequencing is performed to provide a point of reference and enable the comparison of data from both RIBO-seq and RNA-seq during data analysis. Before cells harvesting, you can optionally add chloramphenicol to the bacterial culture, to the final concentration of 100 micrograms per milliliter, to inhibit translation.

Incubate the culture with the antibiotic for one minute. This step is important if harvesting takes longer than usual, as translation inhibition allows to extend sample collection time. Collect the samples with a pre-warmed filtration system.

You can introduce shaking in order to mimic growth conditions. Stop filtering when all media has passed through the membrane, but do not allow the filter to dry completely. Collect bacterial pellets by rapidly scraping the cells off of the filter disc using a pre-warmed, disinfected scoopula.

Immediately place the entire scoopula with the harvested cells in 50 milliliter tube filled with liquid nitrogen. The harvested pellet should be completely covered in liquid nitrogen. Let the pellet freeze thoroughly, and dislodge frozen cells using a previously-cooled scoopula.

Close the lid, and make sure that it's punctured. This is important, as liquid nitrogen can cause closed containers to explode due to the pressure change upon evaporation. Prepare GMPP, DNA swab, and fresh Eppendorf tubes dedicated for lysase.

Prepare lysis buffer with the addition of chloramphenicol if needed. Allocate 500 microliters of lysis buffer per sample into separate Eppendorf tubes, and place them on ice. Decontaminate mortar and pestle with a laboratory disinfectant and 70%ethanol.

Cool mortar and pestle by pouring liquid nitrogen into the mortar. Transfer the frozen bacterial pellet into a pre-chilled mortar and grind it to a powder. Add approximately one volume of aluminum oxide and continue grinding.

Keep mortar, pestle, and the cells cool by pouring liquid nitrogen when needed, to not let the contents of the mortar to thaw. Just before using, add GMPPNP and DNA swab into the lysis buffer aliquot. Transfer the solution into the mortar and continue grinding.

Let the lysase thaw slowly while grinding. When the lysase thaws completely, transfer the mixture into the pre-chilled Eppendorf tube and return to ice. Centrifuge lysase at 20, 0000 G for five minutes at four Celsius degrees.

Transfer supernatants into fresh, pre-chilled Eppendorf tubes and place them on ice. Measure the RNA concentration in each diluted sample with NanoDrop. Divide each lysate into two portions, 0.5 to 1 milligram of RNA for RIBO-seq, and the rest for RNA-seq.

To one milligram of the RNA, add 3.8 microliters of MNase in Tris pH 8, and top it up with lysis buffer to the total volume of 500 microliters. Incubate at 25 Celsius degrees, 300 rounds per minute, for 45 minutes. When incubation is finished, clean the samples with a commercially available RNA clean-up kit.

Prepare 15%polyacrylamide TBE gel with eight molar urea, and place it in a tank with TBE buffer. Pre-run for a minimum of 10 minutes at a constant voltage of 200 volts. Mix the samples with TBE-Urea sample buffer.

Denature at 95 Celsius degrees for one minute and place them immediately on ice. Wash out the urea by injecting TBE buffer into gel wells using a syringe. Load the samples, leaving one well space between them to separate each sample and prevent cross-contamination.

Use 29 nucleotides-long oligo, and a mix of 26 and 32 nucleotides-long oligos as markers. Run the electrophoresis at a constant voltage of 180 volts. Prepare sterile buffer for overnight incubation.

Once the electrophoresis is finished, stain the gel with SYBR Gold. Excise fragments of the gel between 26 and 32 nucleotides with a sterile needle or a razor blade, and place the gel fragments in separate Eppendorf tubes. Change the needle or razor blade between the samples.

Add 200 microliters of overnight incubation buffer to each Eppendorf and incubate the samples at 10 Celsius degrees, 1000 rounds per minute, overnight. Next day, clean the samples with RNA clean-up kit, and elute them in 18 microliters of nuclease-free water. The obtained ribosomal footprints are ready for library preparation.

Perform ribosomal RNA depletion for samples for RNA-seq using a commercially-available kit. Next, clean the samples with RNA clean-up kit. Perform alkaline hydrolysis as follows;prepare the alkaline hydrolysis buffer, add one volume of the buffer to one volume of the sample, and incubate at 95 Celsius degrees for 25 minutes.

Add five microliters of 3 Molar sodium acetate pH 5.5 to each sample in order to stop the reaction. Clean the samples with RNA clean-up kit, and elute them in 18 microliters of nuclease-free water. The obtained randomly fragmented RNA is ready for library preparation.

Add 10 microliters of 10x reaction buffer and five microliters of T4 polynucleotide kinase to each sample. Incubate at 37 Celsius degrees for one and a half hour. After this time, add three microliters of one millimolar ATP and incubate at 37 Celsius degrees for one hour.

Next, clean the samples with RNA clean-up kit. Perform library preparation using the commercially-available kit, according to the protocol provided here. Perform PAGE using 6%polyacrylamide gel.

Stain the gel with SYBR Gold. Excise gel fragments containing the libraries. For RNA-seq samples, the library is between 135 and 180 nucleotides, and for RIBO-seq, between 135 and 170 nucleotides.

Use sterile needles or razor blades and place the excised gel fragments in separate Eppendorf tubes. Remember to change the needle or razor blade between the samples. Add 100 microliters of nuclease-free water to each excised gel fragment.

And incubate them at 10 Celsius degrees, 450 rounds per minute, overnight. The next day, clean the samples with DNA clean-up kit. The obtained libraries are ready for quality and quantity control with high sensitivity, DNA on-chip electrophoresis, and then for next-generation sequencing.

The resulting cDNA libraries present an appropriate quantity and quality required for next-generation sequencing, as verified by high sensitivity, DNA on-chip electrophoresis. The bands and peaks representing the RIBO-seq libraries are more narrow and better defined, compared to this representing the RNA-seq libraries. According to the on-chip electrophoresis, the libraries have the expected lens.

The additional peaks closer to 200 base pairs present in RIBO-seq libraries may indicate hibernating ribosomes, not completely digested ribosomal footprints, or artifacts, for example, rRNA, and can be discarded during bioinformatic analysis when the data obtained from sequencing is trimmed and rRNA tRNA is filtered. The average quantity of cDNA generated in library preparation is 32 nanograms, providing sufficient quantity of material required for next-generation sequencing. After quality control by on-chip electrophoresis, libraries were pulled for single and 50 base pair sequencing on a Illumina's NextSeq 500 platform.

Trimming the adapters and poor-quality sequences resulted in 25 to 47 million reads per sample for RNA-seq samples, and 25 to 50 million reads per sample for RIBO-seq samples. The obtained data was quality control checked with FastQC. Both RNA-seq and RIBO-seq samples presented very good quality.

Mapping of the libraries prepared according to the described protocol yielded 2.4 to 9.6 million of uniquely-mapped reads per sample for RNA-seq samples, and 2.3 to 10.4 million of uniquely-mapped reads for RIBO-seq samples. RIBO-seq data exhibits tripled periodicity and a tall, narrow peak, corresponding to the initiating ribosomes and characteristic of the ribosomal footprints, which is not observed in the RNA-seq data. Moreover, the plot showing the profiles of average coverage of ribosomal footprints and mRNA fragments on the coding sequences reveal the higher proportion of reads mapped to the CDS's in the RIBO-seq data set, as expected.

The visualization of the mapped ribosomal footprints showed very similar patterns compared to the results obtained from the same experiment performed accordingly to the standard RIBO-seq procedure, which includes monosome recovery by sucrose gradient ultracentrifugation. Our protocol has been used to investigate translation regulation in various growth conditions, but can be applied to study other aspects of translation, like detection of translation initiation sites and novel protein coding genes. Sequencing of the libraries prepared according to our guidelines results in sufficient data for comprehensive bioinformatic analysis.

The protocol we present here is quick, easy, and cost-effective, and can be performed with standard laboratory equipment.