TY  - JOUR
AU  - Koubek, Jiří
AU  - Jetzinger, Katharina
AU  - Dror, Shiran
AU  - Irastortza-Olaziregi, Mikel
AU  - Frank, Dana
AU  - Kotan, Ilgin
AU  - Santos, Jaime
AU  - Tippmann, Frank
AU  - Lafrenz, Pascal
AU  - Kaessmann, Henrik
AU  - Amster-Choder, Orna
AU  - Bukau, Bernd
AU  - Kramer, Günter
TI  - A simple, fast, and cost-efficient protocol for ultra-sensitive ribosome profiling.
JO  - Nucleic acids research
VL  - 53
IS  - 17
SN  - 0305-1048
CY  - Oxford
PB  - Oxford Univ. Press
M1  - DKFZ-2025-01982
SP  - gkaf902
PY  - 2025
N1  - DKFZ-ZMBH Alliance / 
AB  - Ribosome profiling has become an essential tool for studying messenger RNA (mRNA) translation in cells with codon-level resolution. However, its widespread application remains hindered by the labour-intensive workflow, low efficiency, and high costs associated with sequencing sample preparation. Here, we present a new cost-effective and ultra-sensitive library preparation method that significantly advances the applicability of ribosome profiling. By implementing bead-coupled enzymatic reactions and product purifications, our approach increases both yield and throughput while maintaining high reproducibility. Demonstrating the sensitivity of the protocol, we prepared libraries from as little as 12 fmol of RNA, which expands the feasibility of ribosome profiling from minimal input samples, such as those derived from small populations, stressed cells, or patient-derived specimens. Additionally, we validate the versatility of the protocol across multiple species and demonstrate its applicability for RNA-seq library preparation. Altogether, this protocol provides a highly accessible and efficient alternative to existing ribosome profiling workflows, facilitating research in previously challenging experimental contexts.
KW  - Ribosomes: metabolism
KW  - Ribosomes: genetics
KW  - RNA, Messenger: genetics
KW  - RNA, Messenger: metabolism
KW  - Humans
KW  - Gene Library
KW  - Protein Biosynthesis
KW  - Animals
KW  - Sequence Analysis, RNA: methods
KW  - Sequence Analysis, RNA: economics
KW  - High-Throughput Nucleotide Sequencing: methods
KW  - Ribosome Profiling
KW  - RNA, Messenger (NLM Chemicals)
LB  - PUB:(DE-HGF)16
C6  - pmid:40966508
C2  - pmc:PMC12448852
DO  - DOI:10.1093/nar/gkaf902
UR  - https://inrepo02.dkfz.de/record/304991
ER  -