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

We published a major update of the nanoCAGE protocol in an extensive book chapter (53 pages!) in Methods in Molecular Biology volume 1543 (see reference to Poulain et al., at the bottom of this page). The biggest changes, illustrated below, are the introduction of unique molecular identifiers in the 5′ linker, and the replacement of the "library" PCR by a fragmentation and amplification step using the standard "tagmentation" kit from Illumina.

The tagmentation step introduces linkers that prepare for multiplexing and sequencing on the Illumina platform. Whole-genome and whole-transcriptome analysis methods use this kit extensively. To adapt it for nanoCAGE, we replaced one of the amplification primers by a custom primer matching our template-switching oligonucleotide, so that only the 5′ fragments will be amplified.

The book chapter includes a 3-page bench workflow summary (see below), which is also available for download from GitHub.