RNA Antisense Purification RAP isolates lncRNAs and maps the sequence of their target DNA through a probe-capture mechanism (Engreitz et al., 2013). First, the cells are crosslinked and lysed before DNase I chromatin digestion to 100_300 bp DNA fragments. Biotinylated RNA probes, antisense to the […]

Precision Nuclear Run-on Sequencing Precision nuclear run-on sequencing (PRO-Seq) maps RNAPII pause sites with base-pair resolution during RNA transcription (Kwak et al., 2013) (Mahat et al., 2016). This approach is similar to GRO-Seq, but it provides the added benefit of single-base resolution. RNAPII initiation sites […]

Precision Nuclear Run-on Sequencing for RNA Polymerase II Start Sites PRO-cap maps RNAPII initiation sites during RNA transcription with base-pair resolution. This approach is a variation of the PRO-Seq method, which maps RNAPII pause sites (Kwak et al., 2013). A nuclear run-on reaction with biotin-NTP […]

Paired-end Analysis of Transcription Start Sites PEAT characterizes transcription start sites (TSS) in mRNA using a technique similar to TIF-Seq (Ni et al., 2010). First, poly(A)+ RNAs are enriched from total RNA and the caps are removed with TAP. The 5′ ends of uncapped mRNAs […]

Parallel Analysis of RNA Ends Sequencing Various RNA degradation processes impart characteristic sequence ends. By analyzing the cleavage sites, the degradation processes can be inferred (German et al., 2008). In PARE-Seq, the degraded uncapped mRNA is ligated to 5′ adapters containing an MmeI restriction site […]

Poly(A)-Tail Length Profiling by Sequencing PAL-Seq measures poly(A)-tail length by incorporating fluorescent tags on biotinylated deoxyuridine triphosphate (dUTPs) and using signal intensity to quantify poly(A)-tail length (Subtelny et al., 2014). Similar to 3P-Seq RNA library preparation, a splint oligonucleotide containing a 3′-adapter sequence is ligated […]

Native Elongating Transcript Sequencing NET-Seq detects nascent, actively transcribed RNAPII RNAs, through the capture of 3′ RNA (Churchman et al., 2011). In this method, the RNAPII elongation complex is immunoprecipitated, and RNA is extracted and reverse-transcribed to cDNA. Deep sequencing of the cDNA allows for […]

Global Run-on Sequencing GRO-Seq maps the binding sites of transcriptionally active RNA polymerase II (RNAPII) (Core et al., 2008). In this method, active RNAPII is allowed to run on in the presence of 5-bromouridine 5′-triphosphate (Br-UTP). RNAs are hydrolyzed and purified using beads coated with […]

RNA-Seq Libraries from Stochastically Terminated 3′-azido-blocked cDNA Fragments ClickSeq is an RNA sequencing technique that uses bioconjugation as an alternative to fragmentation in the library preparation step, to produce lower error rates than standard sequencing methods (Routh et al., 2015). First, RNA is reverse-transcribed into […]

Chromatin Isolation by RNA Purification ChIRP, also commonly referred to as ChIRP-seq, is a protocol to detect the locations on the genome where ncRNAs, such as lncRNAs, and their proteins are bound (Chu et al., 2011). In this method, samples are first crosslinked and sonicated. […]