Abstract
Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). Accurate detection of TSSs enhances identification and discovery of core promoters. In addition, active enhancers can be detected through signatures of bidirectional transcription initiation. Described here is a protocol for performing super-low input carrier-CAGE (SLIC-CAGE). This SLIC adaptation of the CAGE protocol minimizes RNA losses by artificially increasing the RNA amount through use of an in vitro transcribed RNA carrier mix that is added to the sample of interest, thus enabling library preparation from nanogram-amounts of total RNA (i.e., thousands of cells). The carrier mimics the expected DNA library fragment length distribution, thereby eliminating biases that could be caused by the abundance of a homogenous carrier. In the last stages of the protocol, the carrier is removed through degradation with homing endonucleases and the target library is amplified. The target sample library is protected from degradation, as the homing endonuclease recognition sites are long (between 18 and 27 bp), making the probability of their existence in the eukaryotic genomes very low. The end result is a DNA library ready for next-generation sequencing. All steps in the protocol, up to sequencing, can be completed within 6 days. The carrier preparation requires a full working day; however, it can be prepared in large quantities and kept frozen at -80 °C. Once sequenced, the reads can be processed to obtain genome-wide single-nucleotide resolution TSSs. TSSs can be used for core promoter or enhancer discovery, providing insight into gene regulation. Once aggregated to promoters, the data can also be used for 5'-centric expression profiling.
Highlights
Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution genome-wide mapping of RNA polymerase II transcription start sites (TSSs)[1]
To enable TSS profiling using only nanograms of total RNA, we have recently developed Super-low Input Carrier-CAGE10 (SLIC-CAGE, Figure 1)
As the protocol has multiple steps, continuous sample loss will lead to unsuccessful libraries
Summary
Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution genome-wide mapping of RNA polymerase II transcription start sites (TSSs)[1]. The recent CAGE protocol, nAnT-iCAGE11, is the most unbiased protocol for TSS profiling, as it avoids cutting the fragments to short tags using restriction enzymes and does not use PCR amplification. A limitation of the nAnT-iCAGE protocol is the requirement for a large amount of starting material (e.g., 5 μg of total RNA for each sample). If nAnT-iCAGE is successful, only [1,2] ng of DNA library material is available from each sample, thereby limiting the achievable sequencing depth. After specific degradation of the carrier and removal of fragments by size exclusion, the target library is PCR amplified and ready for next-generation sequencing. As SLIC-CAGE is based on the nAnTiCAGE protocol[11], it enables multiplexing of up to eight samples prior to sequencing
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