Froberg JE, Durak O, Macklis JD. 2022. Development of ultra-low-input nanoRibo-seq enables quantification of translational control, revealing broad uORF translation by subtype-specific neurons. BioRxiv. DOI:


While increasingly powerful approaches enable investigation of transcription using small samples of RNA, approaches to investigate translational regulation in small populations of specific cell types, and/or (sub)-cellular contexts are lacking. Comprehensive investigation of mRNAs actively translated into proteins from ultra-low input material would provide important insight into molecular machinery and mechanisms underlying many cellular, developmental, and disease processes in vivo. Such investigations are limited by the large input required for current state-of-the-art Ribo-seq. Here, we present an optimized, ultra-low input “nanoRibo-seq” approach using 102 – 103-fold less input material than standard approaches, demonstrated here in subtype-specific neurons. nanoRibo-seq requires as few as 2.5K neurons, and exhibits rigorous quality control features: 1) strong enrichment for CDS versus UTRs and non-CDS; 2) narrow, distinct length distributions over CDS; 3) ribosome P-sites predominantly in-frame to annotated CDS; and 4) sufficient ribosome-protected fragment (RPF) coverage across thousands of mRNAs. As proof-of-concept, we calculate translation efficiencies from paired Ribo-seq and alkaline fragmented control libraries from “callosal projection neurons” (CPN), revealing divergence between mRNA abundance and RPF abundance for hundreds of genes. Intriguingly, we identify substantial translation of upstream ORFs in the 5’ UTRs of genes involved in axon guidance and synapse assembly. nanoRibo-seq enables previously inaccessible investigation of translational regulation by small, specific cell populations in normal or perturbed contexts.

Related Faculty

Photo of Jeffrey D. Macklis

Jeffrey Macklis investigates molecular controls and mechanisms over neuron subtype specification, development, diversity, axon guidance-circuit formation, and pathology in the cerebral cortex. His lab seeks to apply developmental controls toward brain and spinal cord regeneration and directed differentiation for in vitro mechanistic modeling using human assembloids.

Photo of John Froberg

I use novel sequencing strategies to understand multiple aspects of RNA biology in cortical neurons and growth cones.

Photo of Omer Durak

I am interested in understanding the molecular and cellular mechanisms underlying the establishment of callosal projection neuron (CPN) circuitry, and the role of CPN in disease-relevant social and cognitive behavior.

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