![]() Recent progress in full-length RNA sequencing permitted by single-molecule long-read sequencing platforms (PacBio and Nanopore in particular) has furthered our knowledge on transcriptional regulation by enabling simultaneous detection of elongation and splicing on the same nascent RNA molecule. These methods can be grouped by their strategies for nascent RNA enrichment, including isolating the chromatin fraction (caRNA-seq 11, 12, Start-seq 13, SMIT 4), immunoprecipitation of Pol II (NET-seq 14, mNET-seq 15), nuclear run-on with labeled nucleotide (GRO-seq 16, PRO-seq 17, 18), and metabolic labeling (4sU-seq 19, TT-seq 20, SLAM-seq 21, TimeLapse-seq 22). Many high-throughput sequencing methods have been developed to characterize nascent RNAs at the genome-wide scale quantitatively, and have revealed novel insights into transcriptional regulation by tracking Pol II position at nucleotide resolution as well as the status of splicing 10. Pre-mRNA splicing occurs mainly cotranscriptionally and is tightly coupled with Pol II elongation 1, 2, 3, 4, 5, 6, 7, 8, 9. The cotranscriptional processes, including mRNA capping, splicing, cleavage and polyadenylation, turn nascent RNAs into mature mRNAs that are eventually exported to the cytoplasm. In eukaryotes, transcription by RNA polymerase II (Pol II) involves initiation, elongation and termination. The subsequent long-read sequencing and initial data analysis ranges between 31 and 40 h, depending on the sequencing platform. The minimum time required for FLEP-seq, including RNA extraction and library preparation, is 36 h. We also provide a complete bioinformatic pipeline from raw data processing to downstream analysis. Library production requires as little as one nanogram of nascent RNA (after rRNA/tRNA removal), and either Nanopore or PacBio platforms can be used for sequencing. Here we describe a detailed protocol for this method called FLEP-seq (full-length elongating and polyadenylated RNA sequencing) that is applicable to plants. This method allows calculation of the kinetics of cotranscriptional splicing and detects polyadenylated transcripts with unspliced introns retained at specific positions posttranscriptionally. We recently developed a full-length nascent RNA sequencing method in the model plant Arabidopsis that simultaneously detects RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale. You can hear more from Vincenzo Natali by clicking the link below.Elongation, splicing and polyadenylation are fundamental steps of transcription, and studying their coordination requires simultaneous monitoring of these dynamic processes on one transcript. I believe Natali would be an excellent candidate for a CBM and is someone who understands the genre. It is a modern twist on the Frankenstein tale that pushes the envelope both ethically and sexually. Splice looks to be an amazing movie and is getting rave reviews from its showing at Sundance. He wouldn't mind the same approach being taken with Swamp Thing and the supernatural characters that he came across such as Deadman or John Constantine. You know, turning what they did in the comic book world, which is fully integrated universe of different characters translating that into the film world." "I think what Marvel is doing is so brilliant. We touched base on the possibility of DC bringing together their live action superhero universe: So, that would certainly interest me a great deal." I feel like that there is a great "Judge Dredd" movie that has yet to be made. "I was always strongly disappointed, severely disappointed by "Judge Dredd". ![]() Along with doing an adaptation of DC's Camelot 3000 he touched base on a character that has already been done: We also talked about what other CBM's he would like to make. Once someone buys them, it can be pursued but even then Natali says "it won't be easy." I do find it very interesting that Joel Silver is the producer that bought Splice and is also very interested in bringing Swamp Thing back. The film rights are now with MGM which is in financial ruin. "I have explored what the rights issues are and they're a bit complicated" One of the biggest hurdles would be obtaining the rights to the character: That, for me, is the version that would interest me most, personally." "He really kind of pushed it in this kind of strange philosophical, ecological direction and took the character and, kind of, completely reinvented what the essence of that character is. When I asked him to expand on what drew him to Alan Moore's version:
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