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

BMC Genomics
Yang SongRonna Hertzano

Abstract

Cell type-specific ribosome-pulldown has become an increasingly popular method for analysis of gene expression. It allows for expression analysis from intact tissues and monitoring of protein synthesis in vivo. However, while its utility has been assessed, technical aspects related to sequencing of these samples, often starting with a smaller amount of RNA, have not been reported. In this study, we evaluated the performance of five library prep protocols for ribosome-associated mRNAs when only 250 pg-4 ng of total RNA are used. We obtained total and RiboTag-IP RNA, in three biological replicates. We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA. An additional set of samples was processed using the TruSeq kit with 70 ng, as a 'gold standard' control and the SMART-Seq v4 with 250 pg of total RNA. TruSeq-processed samples had the best metrics overall, with similar results for the 4 ng and 70 ng samples. The results of t...Continue Reading

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Citations

Jun 7, 2019·Scientific Reports·Hope KronmanEric J Nestler
Jun 25, 2020·The Laryngoscope·Ronna HertzanoMaggie S Matern
Sep 19, 2019·Scientific Reports·Dimitra SarantopoulouGregory R Grant
Dec 29, 2020·Behavioural Brain Research·Pei X LuoBrian C Trainor
Jan 26, 2021·Frontiers in Oncology·Xiaofeng MuYe Wang
Dec 31, 2020·Clinical and Translational Medicine·Elisabeth StrackAndreas Weigert
Mar 23, 2021·Frontiers in Plant Science·Lucia BarraClara Conicella
Jun 11, 2021·Journal of Hematology & Oncology·Yalan LeiSi Shi
Apr 22, 2021·American Journal of Physiology. Cell Physiology·Peter Z Schall, Keith E Latham
Sep 7, 2019·Journal of Molecular Biology·Yiwen ZhouYing Wan

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Datasets Mentioned

BETA
GSE104213

Methods Mentioned

BETA
Fluorescence
FACS
RNA-seq
immunoprecipitation
PCR
chip
Illumina sequencing
Ribo-IP

Software Mentioned

HTSeq
ComplexHeatmap
ggplot2
NuG
RiboTag
SMARTer®
NEB
FASTX Toolkit
RiboTag IP
Cufflink

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