Protocol for ribosome-protected fragment isolation from rare leukemia cell populations.

Malignant cells under chemotherapy stress alter protein translation, necessitating sensitive methods to profile rare, surviving populations in vivo. We present a protocol for profiling low-input acute myeloid leukemia samples using an optimized ribosome profiling approach. We describe steps for lysing cell inputs, digesting unshielded RNA, enriching ribosome footprints, and gel-based size selection. We then detail procedures for constructing sequencing-ready libraries and using a computational pipeline to align and quantify both protected fragments and matching transcriptomes for reproducible translation analysis. For complete details on the use and execution of this protocol, please refer to Mayerhofer et al.1.