Low Quantity single strand CAGE (LQ-ssCAGE) maps regulatory enhancers and promoters

Abstract

The Cap Analysis of Gene Expression (CAGE) is a powerful method to identify the Transcription Start Sites (TSSs) of capped RNAs while simultaneously measuring transcripts expression level. CAGE allows mapping at single nucleotide resolution at all active promoters and enhancers. Large CAGE datasets have been produced over the years from individual laboratories and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation Of the Mammalian Genome (FANTOM). These datasets constitute open resource for TSS annotations and gene expression analysis. Here we provide an experimental protocol for the most recent CAGE method called Low Quantity (LQ) single strand (ss) CAGE “LQ-ssCAGE”, which enables cost-effective profiling of low quantity RNA samples. LQ-ssCAGE is especially useful for samples derived from cells cultured in small volumes, cellular compartments such as nuclear RNAs or for samples from developmental stages. We demonstrate the reproducibility and effectiveness of the method by constructing 240 LQ-ssCAGE libraries from 50 ng of THP-1 cell extracted RNAs and discover lowly expressed novel enhancer and promoter-derived lncRNAs.

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification

“…Mouse datasets also contained 100 million Illumina read pairs, but equal amounts of PacBio CCS and ONT dRNA reads were generated (20 million sequences each).To allow users to simulate their own data, the methods described above are implemented as simple command-line scripts which are available at https://github.com/LRGASP/lrgaspsimulation/.CAGE data of WTC-11 samples for validation of transcript 5' endsCAGE data from WTC-11 samples are being produced for validation of transcript 5' ends; therefore, will not be released until the close of the challenge submissions. CAGE data will be obtained from two RNA biological replicates of WTC-11, from the same exact RNA used for long-read sequencing.The 15 µg of WTC-11 RNAs from each biological replicate, ENCODE BioSample Accession #ENCBS944CBA and #ENCBS474NOC, were used for the single strand (ss)CAGE library preparation described in the published protocol24 . Briefly, the 15 µg RNAs were aliquoted to 5 µg in three tubes and reverse transcribed to cDNAs with random primers, and the RNA-cDNA hybrids were cap-trapped by the streptavidin beads.…”

Antisense oligonucleotide-mediated perturbation of long non-coding RNA reveals functional features in stem cells and across cell types

“…HT-CAGE allows samples to be highly-multiplexed by introducing additional barcodes in the RT primer, 22 which is ideal for large-scale perturbation studies. From the POU5F1 knockdown using either ASO or siRNA, we compared the performance of standard nAnTi-CAGE 21 with HT-CAGE, which resulted in less sequencing reads ( Fig.…”

“…To investigate the effect of lncRNA knockdown on gene expression profile (i.e. molecular phenotype), we performed Cap Analysis of Gene Expression (CAGE) 21,22 after knockdown with selected effective ASOs (n = 317; targeting 123 lncRNAs). For each ASO, we performed differential gene expression, pathway enrichment and differential transcription factor (TF) binding motif analyses, comparing against the NC ASO background (Methods).…”

“…Fifty nanogram of purified RNA was used from each sample, which was 96-multiplexed for constructing one single-strand CAGE library, 22 according to an improved version of nAnT-iCAGE protocol 21 that avoids any bias from PCR amplification. Libraries were subjected to 50-base pair-end sequencing using an Illumina HiSeq 2500 instrument.…”

News from around the RNA world: new avenues in RNA biology, biotechnology and therapeutics from the 2022 SIBBM meeting

“…Pietro Carninci (Human Technopole, Milan – Italy and Riken Institute, Japan) is a major expert on lncRNAs and their regulation. His group developed the cap analysis gene expression (CAGE) tool that has the dual goal to identify the transcription start sites (TSSs) of capped RNAs and measure transcripts expression levels ( Maquat et al, 2022 ; Takahashi et al, 2021 ). This powerful tool manages to detect the promoters and enhancers of the transcripts at a single nucleotide resolution.…”