NanoCAGE: A Method for the Analysis of Coding and Noncoding 5′-Capped Transcriptomes

Abstract

Transcripts in all eukaryotes are characterized by the 5'-end specific cap structure in mRNAs. Cap Analysis Gene Expression or CAGE makes use of these caps to specifically obtain cDNA fragments from the 5'-end of RNA and sequences those at high throughput for transcript identification and genome-wide mapping of transcription start sites for coding and noncoding genes. Here, we provide an improved version of our nanoCAGE protocol that has been developed for preparing CAGE libraries from as little as 50 ng of total RNA within three standard working days. Key steps in library preparation have been improved over our previously published protocol to obtain libraries having a good 5'-end selection and a more equal size distribution for higher sequencing efficiency on Illumina MiSeq and HiSeq sequencers. We recommend nanoCAGE as the method of choice for transcriptome profiling projects even from limited amounts of RNA, and as the best approach for genome-wide mapping of transcription start sites within promoter regions.

Optimized protocol for the hepatic differentiation of induced pluripotent stem cells in a fluidic microenvironment

“…NanoCAGE libraries were generated and sequenced as previously described (Poulain et al, 2017). Total RNAs were extracted from samples using Trizol™ Reagent and PureLink RNA Mini Kit (Thermo Fisher Scientific) following the manufacturer's instructions.…”

Zebrafish embryonic tissue differentiation is marked by concurrent cell cycle dynamic and gene promoter regulatory changes

“…NanoCAGE libraries were generated following a protocol described in (24). Pooled libraries, representing FACS sorted populations in triplicate were sequenced with the Illumina HiSeq 2500 50 cycles single-read run operation program, following manufacturer's protocol.…”

Machine-driven parameter optimisation of biochemical reactions

“…RT products were harvested with a multichannel pipette, pooled and purified using Ampure XP beads (Beckman Coulter). Purified pools of single cell RT products were processed for nanoCAGE library preparation (Poulain et al, 2017). Pools of reactions performed using standard primer concentrations with SusperScript III or SuperScript IV; and using alternate primer concentrations with SuperScript IV were subsequently tagged by specific index sequences (Nextera XT DNA Library Preparation Kit, Illumina), pooled and sequenced on a MiSeq instrument.…”

“…The proliferation of protocols using clearly different reaction parameters (Table 1) suggests rather that optimal oligonucleotide concentrations vary with other parameters such as enzyme type or substrate amounts. In the past decade, we have developed a method for quantitative high-throughput gene expression analysis, nanoCAGE (Plessy et al, 2010;Poulain et al, 2017), in which a key step is a templateswitching reverse-transcription. The desired product of a nanoCAGE reaction is a set of sequence reads that align in gene promoters at transcription start sites in proportional quantities to a gene's expression level.…”