A rapid protocol for ribosome profiling of low input samples

Abstract

Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected by ribosomes from nucleolytic digestion. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of RNA. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling.

Is there a localized role for translational quality control?

“…The use of elongation inhibitors (Aviner 2020) is an important tool for imaging when applied with an awareness of the limits on what such techniques can accomplish (Enam et al 2020;Hobson et al 2020). In addition, new low-input methods of ribosome footprinting are promising for bringing the power of the ribosome profiling approach to low-abundance tissues that are challenging to work with, such as neurons (Hornstein et al 2016;Clamer et al 2018;Li et al 2022a;Ferguson et al 2023;Meindl et al 2023), and even single cells (Ozadam et al 2023). Other approaches (Gonzalez et al 2014;Clamer et al 2021) for isolating active ribosomes or ribosomes from particular tissues will also be important for accomplishing these goals (Silva et al 2022).…”

Ribosome Profiling in the Model Diatom Thalassiosira pseudonana

“…Different methods have been used in ribosome profiling studies to isolate digested monosomes, the most prevalent ones being ultracentrifugation via sucrose gradient and sucrose cushion (e.g., Ingolia et al., 2012; Meindl et al., 2023). As described in the Alternate Protocol, density gradient centrifugation was performed to identify optimal nuclease digestion conditions for T. pseudonana which had not been established before.…”

“…More recently, single‐pot reactions have been introduced that exploit the template switching activity of reverse transcriptase (Ferguson et al., 2023; Ozadam et al., 2023). Here, we employ a recently developed protocol for sequencing library preparation that is particularly suited for low input samples (Meindl et al., 2023) and hence for the processing of samples derived from T. pseudonana that can have a low yield. In brief, for sequencing library preparation, an adapter sequence is ligated to the ribosome protected fragments, followed by reverse transcription, circularization of the cDNA and PCR amplification.…”

“…Here, we report the development of a ribosome profiling protocol for the model diatom T. pseudonana CCMP1335, representing the first application of this technique for any marine algae. Previously developed protocols were adapted (McGlincy & Ingolia, 2017; Meindl et al., 2023; Mohammad et al., 2019), which involved optimizations of harvesting strategy and cell lysis conditions, the amount of nuclease for mRNA digestion, and the implementation of a subtractive hybridization step for rRNA removal. Our optimized strategy ensures the generation of high‐quality footprint data for this important class of organisms.…”