Cold Spring Harbor Protocols Journal Impact Factor & Information

Publisher: Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press

Journal description

Cold Spring Harbor Laboratory is renowned for its teaching of biomedical research techniques. For decades, participants in its celebrated, hands-on courses and users of its laboratory manuals have gained access to the most authoritative and reliable methods in molecular and cellular biology. Now that access has moved online. Cold Spring Harbor Protocols is a definitive, interactive source of new and classic research techniques. The database is fully searchable by keyword and subject, and it has many novel features - such as discussion forums and personal folders - made possible by online publication. Its coverage includes cell and molecular biology, genetics, bioinformatics, protein science, and imaging. Protocols are presented step-by-step and edited in the style that has made Molecular Cloning, Antibodies, Cells and many other CSH manuals essential to the work of scientists worldwide. Protocols will be continuously expanded, updated, and annotated by the originators and users of the techniques. CSH Protocols - continuing Cold Spring Harbor Laboratory's 60-year tradition as a source of trusted techniques.

Current impact factor: 4.63

Impact Factor Rankings

Additional details

5-year impact 0.00
Cited half-life 6.30
Immediacy index 0.75
Eigenfactor 0.18
Article influence 1.46
Website Cold Spring Harbour Protocols website
Other titles CSH protocols, Protocols, CSH protocols, CSH protocols online
ISSN 1559-6095
OCLC 62938806
Material type Document, Periodical, Internet resource
Document type Internet Resource, Computer File, Journal / Magazine / Newspaper

Publisher details

Cold Spring Harbor Laboratory Press

  • Pre-print
    • Author can archive a pre-print version
  • Post-print
    • Author can archive a post-print version
  • Conditions
    • Author's pre-print on preprint server
    • Author's pre-print must be updated with citation, DOI and link to article upon publication
    • Publisher's version/PDF may be used after 6 months
    • Publisher's version/PDF and Author's post-print on author's personal website, institutional repository, funder's designated repository
    • Authors retain copyright
    • Content automatically sent to PubMed Central after 6 months
    • Publisher copyright and source must be acknowledged
    • Publisher last contacted on 15/07/2013
  • Classification
    ​ green

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: RNA sequencing (RNA-Seq) uses the capabilities of high-throughput sequencing methods to provide insight into the transcriptome of a cell. Compared to previous Sanger sequencing- and microarray-based methods, RNA-Seq provides far higher coverage and greater resolution of the dynamic nature of the transcriptome. Beyond quantifying gene expression, the data generated by RNA-Seq facilitate the discovery of novel transcripts, identification of alternatively spliced genes, and detection of allele-specific expression. Recent advances in the RNA-Seq workflow, from sample preparation to library construction to data analysis, have enabled researchers to further elucidate the functional complexity of the transcription. In addition to polyadenylated messenger RNA (mRNA) transcripts, RNA-Seq can be applied to investigate different populations of RNA, including total RNA, pre-mRNA, and noncoding RNA, such as microRNA and long ncRNA. This article provides an introduction to RNA-Seq methods, including applications, experimental design, and technical challenges. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 04/2015; DOI:10.1101/pdb.top084970
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    ABSTRACT: There are multiple platforms available for whole-exome enrichment and sequencing (WES). This protocol is based on the Agilent SureSelect Human All Exon platform, which targets ∼50 Mb of the human exonic regions. The SureSelect system uses ∼120-base RNA probes to capture known coding DNA sequences (CDS) from the NCBI Consensus CDS Database as well as other major RNA coding sequence databases, such as Sanger miRBase. The protocol can be performed at the benchside without the need for automation, and the resulting library can be used for targeted next-generation sequencing on an Illumina HiSeq 2000 sequencer. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 03/2015; DOI:10.1101/pdb.prot083659
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    ABSTRACT: Multiple platforms are available for whole-exome enrichment and sequencing (WES). This protocol is based on the Illumina TruSeq Exome Enrichment platform, which captures ∼62 Mb of the human exonic regions using 95-base DNA probes. In addition to covering the RefSeq and Ensembl coding sequences, the enriched sequences also include ∼28 Mb of RefSeq untranslated regions (UTR). The protocol can be performed at the benchside without the need for automation, and the resulting library can be used for targeted next-generation sequencing on an Illumina HiSeq 2000 sequencer. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 03/2015; DOI:10.1101/pdb.prot084863
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    ABSTRACT: This article illustrates the use of the Encyclopedia of DNA Elements (ENCODE) resource to generate or refine hypotheses from genomic data on disease and other phenotypic traits. First, the goals and history of ENCODE and related epigenomics projects are reviewed. Second, the rationale for ENCODE and the major data types used by ENCODE are briefly described, as are some standard heuristics for their interpretation. Third, the use of the ENCODE resource is examined. Standard use cases for ENCODE, accessing the ENCODE resource, and accessing data from related projects are discussed. Although the focus of this article is the use of ENCODE data, some of the same approaches can be used with data from other projects. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 03/2015; DOI:10.1101/pdb.top084988
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    ABSTRACT: Multiple platforms are available for whole-exome enrichment and sequencing (WES). This protocol is based on the Roche NimbleGen SeqCap EZ Exome Library SR platform, which enriches for ∼44 Mb of the human exonic regions. The SeqCap system uses 55- to 105-base DNA probes to capture known coding DNA sequences (CDS) from the NCBI Consensus CDS Database, RefSeq, and Sanger miRBase. The protocol can be performed at the benchside without the need for automation, and the resulting library can be used for targeted next-generation sequencing on an Illumina HiSeq 2000 sequencer. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 03/2015; DOI:10.1101/pdb.prot084855
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    ABSTRACT: The availability of high-throughput sequencing has created enormous possibilities for scientific discovery. However, the massive amount of data being generated has resulted in a severe informatics bottleneck. A large number of tools exist for analyzing next-generation sequencing (NGS) data, yet often there remains a disconnect between these research tools and the ability of many researchers to use them. As a consequence, several online resources and communities have been developed to assist researchers with both the management and the analysis of sequencing data sets. Here we describe the use and applications of common file formats for coding and storing genomic data, consider several web-accessible open-source resources for the visualization and analysis of NGS data, and provide examples of typical analyses with links to further detailed exercises. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 02/2015; DOI:10.1101/pdb.top083667
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    ABSTRACT: This protocol introduces the technique of homologous recombination in bacteria to insert a linear DNA fragment into bacterial artificial chromosomes (BACs). Homologous recombination allows the modification of large DNA molecules, in contrast with conventional restriction endonuclease-based strategies, which cleave large DNAs into numerous fragments and are unlikely to permit the precise targeting afforded by recombination-based approaches. The method uses a phage lambda-derived recombination system (using exo, beta, and gam) as well as other enzymatic activities provided by the host (Escherichia coli). In the method described here, a DNA fragment encoding enhanced cyan fluorescent protein is inserted immediately after the start codon of the gene encoding choline acetyltransferase ("ChAT"), the final enzyme in acetylcholine biosynthesis, using homologous recombination between sequences that are present both on the introduced DNA fragment and in the target BAC. The desired recombination products are identified via positive selection for resistance to kanamycin. In principle, the resulting modified BAC could be used to produce transgenic mice that express this fluorescent protein in cholinergic neurons. The approach described here could be used to insert any DNA fragment. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 02/2015; 2015(2):pdb.prot072397. DOI:10.1101/pdb.prot072397
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    ABSTRACT: Poisoned primer extension is primarily used to distinguish between RNAs that are nearly identical in sequence but cannot be distinguished by standard primer extension because they are the same size (e.g., edited vs. nonedited transcripts). It is conceptually identical to the standard primer extension reaction but involves the use of a chain-terminating dideoxynucleotide (the "poison") in the presence of the other three nucleotides. A radioactively labeled primer that hybridizes a short-distance downstream from the "changed" region of interest is extended by reverse transcription into this region of sequence variation. The reactions contain three of the four substrates for extension (e.g., dATP, dGTP, and dTTP) and a chain-terminating dideoxynucleotide (e.g., ddCTP). The extension reaction stops when reverse transcriptase adds a chain-terminating dideoxynucleotide to the template (e.g., it will add ddCTP when it encounters a G in the template sequence). RNAs that differ in sequence at that position will yield different-sized extension products that can be resolved on a denaturing gel. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot080986. DOI:10.1101/pdb.prot080986
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    ABSTRACT: Initiator caspases, including caspase-2, -8, and -9, are activated by the proximity-driven dimerization that occurs after their recruitment to activation platforms. Here we describe the use of caspase bimolecular fluorescence complementation (caspase BiFC) to measure this induced proximity. BiFC assays rely on the use of a split fluorescent protein to identify protein-protein interactions in cells. When fused to interacting proteins, the fragments of the split fluorescent protein (which do not fluoresce on their own) can associate and fluoresce. In this protocol, we use the fluorescent protein Venus, a brighter and more photostable variant of yellow fluorescent protein (YFP), to detect the induced proximity of caspase-2. Plasmids encoding two fusion products (caspase-2 fused to either the amino- or carboxy-terminal halves of Venus) are transfected into cells. The cells are then treated with an activating (death) stimulus. The induced proximity (and subsequent activation) of caspase-2 in the cells is visualized as Venus fluorescence. The proportion of Venus-positive cells at a single time point can be determined using fluorescence microscopy. Alternatively, the increase in fluorescence intensity over time can be evaluated by time-lapse confocal microscopy. The caspase BiFC strategy described here should also work for other initiator caspases, such as caspase-8 or -9, as long as the correct controls are used. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot082552. DOI:10.1101/pdb.prot082552
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    ABSTRACT: Chemical sequencing of RNA relies on the fact that each of the four bases in RNA is susceptible to chemical modification in a different way. In this protocol, end-labeled RNAs are subjected to base-specific chemical modification reactions that make the RNA strand adjacent to the modified base susceptible to cleavage. The chemical modification reaction is base-specific but limited so that not every base in every strand is modified. After cleavage, the resulting set of radioactive fragments is resolved via polyacrylamide gel electrophoresis. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot080937. DOI:10.1101/pdb.prot080937
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    ABSTRACT: Bioluminescence imaging (BLI) has become an essential technique for preclinical evaluation of anticancer therapeutics and provides sensitive and quantitative measurements of tumor burden in experimental cancer models. For light generation, a vector encoding firefly luciferase is introduced into human cancer cells that are grown as tumor xenografts in immunocompromised hosts, and the enzyme substrate luciferin is injected into the host. Alternatively, the reporter gene can be expressed in genetically engineered mouse models to determine the onset and progression of disease. In addition to expression of an ectopic luciferase enzyme, bioluminescence requires oxygen and ATP, thus only viable luciferase-expressing cells or tissues are capable of producing bioluminescence signals. Here, we summarize a BLI protocol that takes advantage of advances in hardware, especially the cooled charge-coupled device camera, to enable detection of bioluminescence in living animals with high sensitivity and a large dynamic range. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot078261. DOI:10.1101/pdb.prot078261
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    ABSTRACT: Fluorescence recovery after photobleaching (FRAP) is a microscopy technique for measuring the kinetics of fluorescently labeled molecules and can be applied both in vitro and in vivo for two- and three-dimensional systems. This introduction discusses the three basic FRAP methods: traditional FRAP, multiphoton FRAP (MPFRAP), and FRAP with spatial Fourier analysis (SFA-FRAP). Each discussion is accompanied by a description of the mathematical analysis appropriate for situations in which the recovery kinetics is dictated by free diffusion. In some experiments, the recovery kinetics is dictated by the boundary conditions of the system, and FRAP is then used to quantify the connectivity of various compartments. Because the appropriate mathematical analysis is independent of the bleaching method, the analysis of compartmental connectivity is discussed last, in a separate section. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.top083519. DOI:10.1101/pdb.top083519
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    ABSTRACT: The use of magnetic resonance imaging (MRI) in humans and animal models has been rapidly growing. MRI provides high spatial resolution, excellent tissue contrast, and outstanding definition of the anatomical structure of normal organs and tumors. Because MRI does not require genetically encoded reporters, it can be used for tumor surveillance and the assessment of treatment effects in a variety of mouse cancer models. MRI systems for preclinical imaging typically operate at higher magnetic field strength, ranging from 4.7 to 15 T, as opposed to clinical MRI scanners, which range from 1.5 to 3 T. The higher field strength of dedicated preclinical systems provides higher spatial resolution and higher signal-to-noise ratios. MRI of mouse cancer models requires optimization of numerous parameters, including pulse sequences and radio frequency coils. Here, we describe a protocol covering the general procedures for MRI. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot078253. DOI:10.1101/pdb.prot078253
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    ABSTRACT: Genetically encoded, ratiometric, fluorescent Ca(2+) biosensors can be used in living cells to quantitatively measure free Ca(2+) concentrations in the cytosol or in organelles. This protocol describes how to perform a calibration of a Ca(2+) sensor expressed in cultured mammalian cells as images are acquired using a widefield fluorescence microscope. This protocol also explains how to calculate Förster resonance energy transfer (FRET) ratios from acquired images and how to convert FRET ratios to Ca(2+) concentrations. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot076547. DOI:10.1101/pdb.prot076547
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    ABSTRACT: Conventional liquid chromatography on phosphocellulose (PC) can be used to separate tubulin and microtubule-associated proteins (MAPs). Tubulin is a highly acidic protein and thus does not bind to PC. MAPs, however, do bind to PC and can be eluted with a subsequent salt wash of the column. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot081208. DOI:10.1101/pdb.prot081208
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    ABSTRACT: The field of single-cell analysis has greatly benefitted from recent technological advances allowing scientists to study genomes, transcriptomes, proteomes, and metabolomes at the single-cell level. Transcriptomics allows a unique window into cell function and is especially useful for studying global variability among single cells of seemingly the same type. Generating transcriptome data from RNA samples has become increasingly easy and can be done using either microarray or RNA-Seq techniques. RNA isolation is the first step of transcriptomics. Numerous RNA isolation procedures exist and differ with respect to the type and number of cells from which they are capable of isolating RNA. Although it is trivial to isolate RNA from bulk tissue or culture plates, sophisticated methods are required to capture RNA from single cells in a pool of cells or in intact tissue. We describe here the protocols used for isolating the soma of single neurons in cultures and in tissue slices using the pipette capture and the PALM or laser capture microdissection (LCM) approaches, respectively. LCM was developed to isolate cells from tissue sections primarily for pathological tissue analysis. LCM can be used to isolate individual cells or groups of cells from ethanol or paraffin-embedded formalin-fixed tissue sections and dissociated tissue cultures. The soma isolates from either technique can subsequently be used for RNA amplification procedures and transcriptome analysis. These procedures can also be adapted to other cell types in cultures and tissue sections and can be used on neuronal subcellular structures, such as dendrites. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot072439. DOI:10.1101/pdb.prot072439
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    ABSTRACT: Förster resonance energy transfer (FRET) occurs across very short distances (in the nanometer range) between donor and acceptor fluorophores that overlap in their emission and absorption spectra. FRET-compatible green fluorescent protein (GFP) variants that are fused to short peptide linkers containing caspase cleavage sites can be used to measure caspase activity. In the intact probes, the donor and acceptor fluorophores are in close proximity, and FRET is highly efficient. On caspase activation, proteolysis of the linker occurs, and the donor is separated from the acceptor. This results in a disruption of resonance energy transfer and an increase in donor fluorescence quantum yield; this event is typically referred to as sensitized emission or donor unquenching. A number of highly sensitive FRET probes based on the cyan fluorescent protein-yellow fluorescent protein (CFP-YFP) pair, or improved variants thereof, have been developed to detect intracellular caspase activities. In this protocol we describe how to use FRET-based caspase substrates and time-lapse imaging to measure caspase activity in cells undergoing apoptosis. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot082560. DOI:10.1101/pdb.prot082560
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    ABSTRACT: In the last 15 years, there has been an explosion in the development of genetically encoded biosensors that report enzyme activity, chemical transformation, or concentration of ions and molecules in living cells. Currently, there are well over 120 biosensors of different cellular targets. As a general design principle, these sensors convert a molecular event, such as the binding of a molecule to a sensing domain or a signal-induced change in protein conformation, into a change in the sensor's fluorescence properties. In contrast to small-molecule sensors, genetically encoded sensors are generated when sensor-encoding nucleic acid sequences, which have been introduced by transgenic technologies, are translated in cells, tissues, or organisms. One of the best developed classes of biosensors is the genetically encoded Ca(2+) indicators (GECIs). Here, we briefly summarize the properties of ratiometric GECIs and describe how they are used to quantify Ca(2+) in specific cellular locations, such as the cytosol, nucleus, endoplasmic reticulum, and mitochondria. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.top066043. DOI:10.1101/pdb.top066043
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    ABSTRACT: Over the last two decades, advances in experimental and computational technologies have greatly facilitated genomic research. Next-generation sequencing technologies have made de novo sequencing of large genomes affordable, and powerful computational approaches have enabled accurate annotations of genomic DNA sequences. Charting functional regions in genomes must account for not only the coding sequences, but also noncoding RNAs, repetitive elements, chromatin states, epigenetic modifications, and gene regulatory elements. A mix of comparative genomics, high-throughput biological experiments, and machine learning approaches has played a major role in this truly global effort. Here we describe some of these approaches and provide an account of our current understanding of the complex landscape of the human genome. We also present overviews of different publicly available, large-scale experimental data sets and computational tools, which we hope will prove beneficial for researchers working with large and complex genomes. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.top083642. DOI:10.1101/pdb.top083642