Capture Hybridization Analysis of RNA Targets CHART maps genomic binding sites of ncRNAs by isolating and sequencing the DNA regions where the crosslinked RNA-DNA-protein complexes are bound (Simon et al., 2011). CHART differs from other crosslinked-complex purification techniques, such as ChIRP, due to the use […]

Cap Analysis Gene Expression Sequencing CAGE measures RNA expression and maps TSS in promoters (Takahashi et al., 2012). In this method, RNA is first reverse-transcribed using random primers. The RNA cap and 3′ ends are biotinylated. Nonhybridized, single-stranded RNAs are digested with RNase, leaving 5′ […]

Bromouridine Sequencing Bru-Seq maps nascent RNA transcripts using bromouridine tagging (Paulsen et al., 2013). Active RNAPII synthesizes RNA in the presence of Br-UTP. Tagged RNA transcripts are immunoseparated from total RNA using magnetic beads coated with anti-BrdU antibodies. The captured RNA transcripts are eluted and […]

Bromouridine 5,6-dichlorobenzimidazole1-b-D-ribofuranoside Sequencing BruDRB-Seq reports the elongation rate of RNAPII (Veloso et al., 2014). 5,6-dichlorobenzimidazole 1-beta-D-ribofuranoside (DRB) is added to cells before elongation to inhibit RNAPII transiently, allowing synchronized transcriptional initiation throughout the genome. Upon removal of DRB, Br-UTP is added instead of UTP, along […]

Bromouridine Pulse-chase Sequencing BruChase-Seq uses bromouridine tagging to map and quantify the relative stability of nascent RNA transcripts (Paulsen et al., 2013). RNAPII synthesizes RNA in the presence of Br-UTP. Untagged uridine is added to chase out bromouridine from RNAs with high turnover. Long-lived RNA […]

5′ Global Run-on Sequencing 5′-GRO-Seq maps the sequences of nascent RNA with a 7-methylguanylate (m7G) cap at any given time using labeled nucleotides (Lam et al., 2013). This method was originally developed to map and detect instabilities in TSS due to the presence of enhancer […]

Quantitatively Measure Abundance of 3’UTR Isoforms 3′-Seq was designed to measure the abundance of 3′-UTR isoforms quantitatively in a wide array of human tissue types (Lianoglou et al., 2013). The first cDNA strand is generated by reverse-transcribing total RNA using an oligo(dT) primer containing a […]

3′-End Sequencing for Expression Quantification 3′-end Sequencing for Expression Quantification (3Seq) is able to isolate highly degraded mRNA from formalin-fixed paraffin-embedded (FFPE) tissue samples for quantitative genome-wide expression analysis (Beck et al., 2010). First, mRNA is isolated from total RNA by poly(A) selection. Next, an […]

Poly(A)-Position Profiling by Sequencing Poly(A)-position profiling by sequencing (3P-Seq) is used to identify 3′-UTRs in mRNA (Jan et al., 2011). Poly(A) selection is used to isolate mRNA from total RNA, and biotinylated-splint primers are annealed and splint-ligated to the end of the mRNA poly(A) tail. […]

3′ End of Nascent Transcripts The 3’NT method sequences nascent RNA transcripts to map the positions of elongating and arrested RNAPII complexes at nucleotide resolution (Weber et al., 2014). This method effectively isolates the RNAPII-chromatin complex by capitalizing on its ability to remain bound to […]