skip to main content

Explore Research Products in the NSF-PAR

Title: Smar2C2: A Simple and Efficient Protocol for the Identification of Transcription Start Sites
Abstract

Promoters and the noncoding sequences that drive their function are fundamental aspects of genes that are critical to their regulation. The transcription preinitiation complex binds and assembles on promoters where it facilitates transcription. The transcription start site (TSS) is located downstream of the promoter sequence and is defined as the location in the genome where polymerase begins transcribing DNA into RNA. Knowing the location of TSSs is useful for annotation of genes, identification of non‐coding sequences important to gene regulation, detection of alternative TSSs, and understanding of 5′ UTR content. Several existing techniques make it possible to accurately identify TSSs, but are often difficult to perform experimentally, require large amounts of input RNA, or are unable to identify a large number of TSSs from a single sample. Many of these protocols take advantage of template switching reverse transcriptases (TSRTs), which reliably place an adaptor at the 5′ end of a first strand synthesis of cDNA. Here, we introduce a protocol that exploits TSRT activity combined with rolling circle amplification to identify TSSs with several unique advantages over existing methods. Sequence adaptors are placed on the 5′ and 3′ end of the full‐length cDNA copy of a transcript. A splint compatible with those adaptors is then used to circularize the full‐length cDNA. Linear DNA containing concatemers of the cDNA are generated using rolling circle amplification, and a sequencing library is formed by fragmenting the concatemers. This protocol is straightforward to execute, requiring limited bench time with relatively stable reagents. Using extremely low amounts of RNA input, this protocol produces large numbers of accurate, deduplicated TSSs genome wide. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Splint generation

Basic Protocol 2: RNA extraction

Basic Protocol 3: cDNA synthesis

Basic Protocol 4: cDNA circularization and amplification

Basic Protocol 5: Library generation

  more » « less
Award ID(s):
1856627
NSF-PAR ID:
10403626
Author(s) / Creator(s):
 ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Current Protocols
Volume:
3
Issue:
3
ISSN:
2691-1299
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ( , Current Protocols in Neuroscience)
    Abstract

    Nanobodies (nAbs) are recombinant antigen‐binding variable domain fragments obtained from heavy‐chain‐only immunoglobulins. Among mammals, these are unique to camelids (camels, llamas, alpacas, etc.). Nanobodies are of great use in biomedical research due to their efficient folding and stability under a variety of conditions, as well as their small size. The latter characteristic is particularly important for nAbs used as immunolabeling reagents, since this can improve penetration of cell and tissue samples compared to conventional antibodies, and also reduce the gap distance between signal and target, thereby improving imaging resolution. In addition, their recombinant nature allows for unambiguous definition and permanent archiving in the form of DNA sequence, enhanced distribution in the form of sequences or plasmids, and easy and inexpensive production using well‐established bacterial expression systems, such as the IPTG induction method described here. This article will review the basic workflow and process for developing, screening, and validating novel nAbs against neuronal target proteins. The protocols described make use of the most common nAb development method, wherein an immune repertoire from an immunized llama is screened via phage display technology. Selected nAbs can then be taken through validation assays for use as immunolabels or as intrabodies in neurons. © 2020 Wiley Periodicals LLC.

    This article was corrected on 26 June 2021. See the end of the full text for details.

    Basic Protocol 1: Total RNA isolation from camelid leukocytes

    Basic Protocol 2: First‐strand cDNA synthesis; VHH and VHrepertoire PCR

    Basic Protocol 3: Preparation of the phage display library

    Basic Protocol 4: Panning of the phage display library

    Basic Protocol 5: Small‐scale nAb expression

    Basic Protocol 6: Sequence analysis of selected nAb clones

    Basic Protocol 7: Nanobody validation as immunolabels

    Basic Protocol 8: Generation of nAb‐pEGFP mammalian expression constructs

    Basic Protocol 9: Nanobody validation as intrabodies

    Support Protocol 1: ELISA for llama serum testing, phage titer, and screening of selected clones

    Support Protocol 2: Amplification of helper phage stock

    Support Protocol 3: nAb expression in amber suppressorE. colibacterial strains

     
    more » « less
  2. ; ( , Genomics, Proteomics & Bioinformatics)
    Abstract

    Endogenously-encoded microRNAs (miRNAs) are a class of small regulatory RNAs that modulate gene expression at the post-transcriptional level. In plants, miRNAs have increasingly been identified by experiments based on next-generation sequencing (NGS). However, promoter organization is currently unknown for most plant miRNAs, which are transcribed by RNA polymerase II. This deficiency prevents a comprehensive understanding of miRNA-mediated gene networks. In this study, by analyzing full-length cDNA sequences related to miRNAs, we mapped transcription start sites (TSSs) for 62 and 55 miRNAs in Arabidopsis and rice, respectively. The average free energy (AFE) profiles in the vicinity of TSSs were studied for both species. By employing position weight matrices (PWM) for 99 plant cis-elements, we discovered that three cis-elements were over-represented in the miRNA promoters of both species, while four and ten cis-elements were over-represented in Arabidopsis only and in rice only. Thus, comparison of miRNA promoters between Arabidopsis and rice provides a new perspective for studying miRNA regulation in plants.

     
    more » « less
  3. ; ( , Microbiology Spectrum)
    Polen, Tino (Ed.)
    ABSTRACT Regulation of gene expression is a vital component of cellular biology. Transcription factor proteins often bind regulatory DNA sequences upstream of transcription start sites to facilitate the activation or repression of RNA polymerase. Research laboratories have devoted many projects to understanding the transcription regulatory networks for transcription factors, as these regulated genes provide critical insight into the biology of the host organism. Various in vivo and in vitro assays have been developed to elucidate transcription regulatory networks. Several assays, including SELEX-seq and ChIP-seq, capture DNA-bound transcription factors to determine the preferred DNA-binding sequences, which can then be mapped to the host organism’s genome to identify candidate regulatory genes. In this protocol, we describe an alternative in vitro , iterative selection approach to ascertaining DNA-binding sequences of a transcription factor of interest using restriction endonuclease, protection, selection, and amplification (REPSA). Contrary to traditional antibody-based capture methods, REPSA selects for transcription factor-bound DNA sequences by challenging binding reactions with a type IIS restriction endonuclease. Cleavage-resistant DNA species are amplified by PCR and then used as inputs for the next round of REPSA. This process is repeated until a protected DNA species is observed by gel electrophoresis, which is an indication of a successful REPSA experiment. Subsequent high-throughput sequencing of REPSA-selected DNAs accompanied by motif discovery and scanning analyses can be used for determining transcription factor consensus binding sequences and potential regulated genes, providing critical first steps in determining organisms’ transcription regulatory networks. IMPORTANCE Transcription regulatory proteins are an essential class of proteins that help maintain cellular homeostasis by adapting the transcriptome based on environmental cues. Dysregulation of transcription factors can lead to diseases such as cancer, and many eukaryotic and prokaryotic transcription factors have become enticing therapeutic targets. Additionally, in many understudied organisms, the transcription regulatory networks for uncharacterized transcription factors remain unknown. As such, the need for experimental techniques to establish transcription regulatory networks is paramount. Here, we describe a step-by-step protocol for REPSA, an inexpensive, iterative selection technique to identify transcription factor-binding sequences without the need for antibody-based capture methods. 
    more » « less
  4. ; ( , Current Protocols in Molecular Biology)
    Abstract

    Long read sequencing technologies now allow high‐quality sequencing of RNAs (or their cDNAs) that are hundreds to thousands of nucleotides long. Long read sequences of nascent RNA provide single‐nucleotide‐resolution information about co‐transcriptional RNA processing events—e.g., splicing, folding, and base modifications. Here, we describe how to isolate nascent RNA from mammalian cells through subcellular fractionation of chromatin‐associated RNA, as well as how to deplete poly(A)+RNA and rRNA, and, finally, how to generate a full‐length cDNA library for use on long read sequencing platforms. This approach allows for an understanding of coordinated splicing status across multi‐intron transcripts by revealing patterns of splicing or other RNA processing events that cannot be gained from traditional short read RNA sequencing. © 2020 Wiley Periodicals LLC.

    Basic Protocol 1: Subcellular fractionation

    Basic Protocol 2: Nascent RNA isolation and adapter ligation

    Basic Protocol 3: cDNA amplicon preparation

     
    more » « less
  5. ; ; ( , Current Protocols in Molecular Biology)
    Abstract

    Base‐editing technologies enable the introduction of point mutations at targeted genomic sites in mammalian cells, with higher efficiency and precision than traditional genome‐editing methods that use DNA double‐strand breaks, such as zinc finger nucleases (ZFNs), transcription‐activator‐like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR‐associated protein 9 (CRISPR‐Cas9) system. This allows the generation of single‐nucleotide‐variant isogenic cell lines (i.e., cell lines whose genomic sequences differ from each other only at a single, edited nucleotide) in a more time‐ and resource‐effective manner. These single‐nucleotide‐variant clonal cell lines represent a powerful tool with which to assess the functional role of genetic variants in a native cellular context. Base editing can therefore facilitate genotype‐to‐phenotype studies in a controlled laboratory setting, with applications in both basic research and clinical applications. Here, we provide optimized protocols (including experimental design, methods, and analyses) to design base‐editing constructs, transfect adherent cells, quantify base‐editing efficiencies in bulk, and generate single‐nucleotide‐variant clonal cell lines. © 2020 Wiley Periodicals LLC.

    Basic Protocol 1: Design and production of plasmids for base‐editing experiments

    Basic Protocol 2: Transfection of adherent cells and harvesting of genomic DNA

    Basic Protocol 3: Genotyping of harvested cells using Sanger sequencing

    Alternate Protocol 1: Next‐generation sequencing to quantify base editing

    Basic Protocol 4: Single‐cell isolation of base‐edited cells using FACS

    Alternate Protocol 2: Single‐cell isolation of base‐edited cells using dilution plating

    Basic Protocol 5: Clonal expansion to generate isogenic cell lines and genotyping of clones

     
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email