A comparative analysis of library prep approaches for sequencing low input translatome samples

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Cell type-specific ribosome pulldown (RiboTag) is valuable for in vivo gene expression analysis.
• Technical challenges with low RNA input for RiboTag sequencing remain underexplored.

Purpose of the Study:

• To evaluate the performance of five library preparation kits for RiboTag-IP samples with limited total RNA input (250 pg–4 ng).
• To identify optimal library preparation methods for RiboTag sequencing with low RNA quantities.

Main Methods:

• Compared five library preparation kits (NuGEN Ovation, TaKaRa SMARTer, TaKaRa SMART-Seq v4, Illumina TruSeq, NEBNext Ultra) using 4 ng of total RNA.
• Utilized 250 pg (SMART-Seq v4) and 70 ng (TruSeq) as additional input amounts for comparison.
• Analyzed RiboTag-IP and total RNA samples in biological triplicates.

Main Results:

• Illumina TruSeq and TaKaRa SMART-Seq v4 kits yielded the highest quality libraries.
• SMART-Seq v4 performed comparably to TruSeq even with significantly lower RNA input (250 pg vs. 4 ng).
• RiboTag-IP samples showed increased intronic reads, indicating capture of immature mRNA; SMARTer kit showed higher non-coding RNA representation.

Conclusions:

• Successful RiboTag-seq is achievable with as little as 250 pg of total RNA using the SMART-Seq v4 kit.
• TruSeq and SMART-Seq v4 kits are recommended for high-quality RiboTag libraries from low RNA inputs.
• RiboTag IP RNA includes immature transcripts, a factor to consider during analysis.