A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol
January 2021
Authors:
Dimitrios G Anastasakis, Alexis Jacob, Parthena Konstantinidou, Kazuyuki Meguro, Duncan Claypool, Pavol Cekan, Astrid D Haase, Markus Hafner
Info:
The authors offer an improvement to the PAR-CLIP method that is more streamlined and does not require the use of radioactive labels. CLIP-seq (CrossLinking and ImmunoPrecipitation) is used to evaluate protein-RNA interactions during gene expression. PAR-CLIP uses in vivo labelling of RNA with photoreactive nucleosides . The method outlined in this protocol (fPAR-CLIP) uses direct ligation of fluorescent adapters to RNA/protein complexes, followed by the isolation of the RNA. The original PAR-CLIP method requires size fractionation on denaturing polyacrylamide gels. The fPAR-CLIP method presented here eliminates this requirement, is more sensitive, and is completed in 2 rather than 4 days.
Pippin Prep is used to size select PCR fragments and eliminate primers and adapters.
Author Affiliations:
Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Disease, NIH, Bethesda MD
Laboratory of Cellular and Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda MD
Laboratory of Clinical Immunology & Microbiology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda MD
MultiplexDX, Bratislava, Slovakia
Citation:
Nucleic Acids Research
DOI: 10.1093/nar/gkab011
isoCirc catalogs full-length circular RNA isoforms in human transcriptomes
January 2021
Authors:
Ruijiao Xin, Yan Gao, Yuan Gao, Robert Wang, Kathryn E. Kadash-Edmondson, Bo Liu, Yadong Wang, Lan Lin & Yi Xing
Info:
The authors present a protocol, isoCirc, for the full-length sequence determination of circular RNAs. The method features rolling circle amplification followed by nanopore sequencing, and includes a description of an integrated computation pipeline. A catalogue of over 100,000 circular RNAs across 12 human tissues and the HEK293 cell line was produced, including ~40,000 isoforms.
BluePippin was used to size select the RCA products after debranching prior to ONT MinION library prep.
Author Affiliations:
Center for Computational and Genomic Medicine, The Children’s Hospital of Philadelphia, PA
Genomics and Computational Biology Graduate Program, University of Pennsylvania
Department of Computer Science and Technology, Center for Bioinformatics, Harbin Institute of Technology, Harbin, China
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA
Citation:
Nature Communications
DOI: 10.1038/s41467-020-20459-8
Identifying the true number of specimens of the extinct blue antelope (Hippotragus leucophaeus)
Jan 2021
Authors:
Elisabeth Hempel, Faysal Bibi, J. Tyler Faith, James S. Brink, Daniela C. Kalthoff, Pepijn Kamminga, Johanna L. A. Paijmans, Michael V. Westbury, Michael Hofreiter & Frank E. Zachos
Info:
In an effort to better understand the mechanisms leading to the extinction of the only large African mammal in modern times, scientists sampled existing museum specimens. These were subject to genomic and mitochondrial sequencing. After using DNA extraction methods designed for ancient specimens, the Pippin Prep was used to exclude DNA fragments (<165 bp) from specimens that yielded very short fragments. This resulted in several-fold more mappable endogenous regions. The authors report that only four of the sixteen specimens were in fact blue antelope and highlight how genetics can be used to identify rare museum species.
Author Affiliations:
Evolutionary Adaptive Genomics, Universität Potsdam, Potsdam Germany
Museum Für Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, Berlin Germany
Natural History Museum of Utah, University of Utah, Salt Lake City UT
Natural History Museum of Utah, University of Utah, Salt Lake City UT
Florisbad Quaternary Research Station and Department, National Museum, Republic of South Africa
Centre for Environmental Management, University of the Free State, Republic of South Africa
Swedish Museum of Natural History, Stockholm, Sweden
Naturalis Biodiversity Center, Leiden, The Netherlands
Section for Evolutionary Genomics, The GLOBE Institute, University of Copenhagen, Denmark
Nature Scientific Reports
ATAC-seq with unique molecular identifiers improves quantification and footprinting
November 2020
Authors:
Tao Zhu, Keyan Liao, Rongfang Zhou, Chunjiao Xia & Weibo Xie
Info:
The authors suggest an improvement on the ATAC-seq method (Assay for Transposase-Accessible Chromatin with high-throughput sequencing). By using unique molecular identifiers to distinguish between transposase insertions and PCR duplicates. They go on to demonstrate the UMI-ATAC-seq method more accurately quantifies chromatin accessibility and improve the sensitivity of identifying transcription footprints.
The PippinHT was used to size select libraries prior to sequencing.
Author Affiliations:
Huazhong Agricultural University, Wuhan China
Citation:
Nature Communications Biology
DOI: 10.1038/s42003-020-01403-4
Complete and haplotype-specific sequence assembly of segmental duplication-mediated genome rearrangements using targeted CRISPR-targeted ultra-long read sequencing (CTLR-Seq)
October 2020
Authors:
Bo Zhou, GiWon Shin, Stephanie U. Greer, Lisanne Vervoort, Yiling Huang, Reenal Pattni, Marcus Ho, Wing H. Wong, Joris R. Vermeesch, Hanlee P. Ji, Alexander E. Urban
Info:
In this preprint the authors report on a method, CTLR-Seq, that combines Sage Science’s HLS-CATCH with low input Oxford Nanopore sequencing(using the MinION platform). The researchers are studying complex and highly repetitive genomic regions associated with neuropsychiatric disorders (mutations at 22q11.2 and at 16p11.2). Long read nanopore sequencing is used to resolve large segmental duplications, copy number variations, and large deletions.
Author Affiliations:
Stanford University, Stanford CA
KU Leuven, Flanders Belgium
Citation:
BioRxiv Preprint
DOI:10.1101/2020.10.23.349621