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{"title"=>"Rapid and unambiguous detection of DNase I hypersensitive site in rare population of cells", "type"=>"journal", "authors"=>[{"first_name"=>"Wei Ping", "last_name"=>"Zeng", "scopus_author_id"=>"56659602600"}, {"first_name"=>"Margaret M.", "last_name"=>"McFarland", "scopus_author_id"=>"56394065800"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pui"=>"372852784", "pmid"=>"24465674", "sgr"=>"84908192095", "scopus"=>"2-s2.0-84908192095", "doi"=>"10.1371/journal.pone.0085740"}, "id"=>"9a78b0c7-fdec-3636-8def-7a71244e6880", "abstract"=>"DNase I hypersensitive (DHS) sites are important for understanding cis regulation of gene expression. However, existing methods for detecting DHS sites in small numbers of cells can lead to ambiguous results. Here we describe a simple new method, in which DNA fragments with ends generated by DNase I digestion are isolated and used as templates for two PCR reactions. In the first PCR, primers are derived from sequences up- and down-stream of the DHS site. If the DHS site exists in the cells, the first PCR will not produce PCR products due to the cuts of the templates by DNase I between the primer sequences. In the second PCR, one primer is derived from sequence outside the DHS site and the other from the adaptor. This will produce a smear of PCR products of different sizes due to cuts by DNase I at different positions at the DHS site. With this design, we detected a DHS site at the CD4 gene in two CD4 T cell populations using as few as 2×10(4) cells. We further validated this method by detecting a DHS site of the IL-4 gene that is specifically present in type 2 but not type 1 T helper cells. Overall, this method overcomes the interference by genomic DNA not cut by DNase I at the DHS site, thereby offering unambiguous detection of DHS sites in the cells.", "link"=>"http://www.mendeley.com/research/rapid-unambiguous-detection-dnase-i-hypersensitive-site-rare-population-cells", "reader_count"=>5, "reader_count_by_academic_status"=>{"Researcher"=>2, "Student > Ph. D. Student"=>1, "Student > Postgraduate"=>1, "Student > Bachelor"=>1}, "reader_count_by_user_role"=>{"Researcher"=>2, "Student > Ph. D. Student"=>1, "Student > Postgraduate"=>1, "Student > Bachelor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>4}, "reader_count_by_subdiscipline"=>{"Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>4}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}}, "reader_count_by_country"=>{"Russia"=>1}, "group_count"=>0}

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Figshare

• {"files"=>["https://ndownloader.figshare.com/files/1352656"], "description"=>"<p>A. Summary of the major steps of the experimental procedure. B. Design of PCR detection of the CD4 DHS site. Short horizontal bars with vertical stripes represent the adaptor sequence. The dark spheres at the end of the adaptors represent biotin. Primer locations are indicated by the horizontal arrows.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "DNA structure", "genetics", "epigenetics", "chromatin", "gene expression", "genomics", "Chromosome biology", "Model organisms", "Animal models", "mouse", "Molecular cell biology"], "article_id"=>905166, "categories"=>["Biological Sciences"], "users"=>["Wei-ping Zeng", "Margaret M. McFarland"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085740.g001", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Method_design_/905166", "title"=>"Method design.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-21 02:58:10"}
• {"files"=>["https://ndownloader.figshare.com/files/1352657"], "description"=>"<p>A. Nuclei isolated from 2×10⁵ total CD4 T cells were treated with the indicated amounts of DNase I. After the treatment, the nuclei were embedded in low-melt agarose gel. Genomic DNA was in-gel purified then released from the gel plug and electrophoresed on a 0.7% agarose gel. B. Left panel shows the isolation of naïve CD4 Tcon cells and nTreg cells by FACS. In the right panel, the nuclei from the isolated cells were treated with 1.25 units of DNase I. In-gel purified genomic DNA was analysed as in A.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "DNA structure", "genetics", "epigenetics", "chromatin", "gene expression", "genomics", "Chromosome biology", "Model organisms", "Animal models", "mouse", "Molecular cell biology"], "article_id"=>905167, "categories"=>["Biological Sciences"], "users"=>["Wei-ping Zeng", "Margaret M. McFarland"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085740.g002", "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_DNase_I_treatment_of_nuclei_/905167", "title"=>"DNase I treatment of nuclei.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-21 02:58:10"}
• {"files"=>["https://ndownloader.figshare.com/files/1352658"], "description"=>"<p>After in-gel purification and blunt-ending, the genomic DNA of naïve CD4 Tcon and nTreg cells were ligated to biotinylated adaptor. The ligated DNA of high molecular weight (>23 kb) was gel purified to remove free adaptors, then fragmented to an average size of 500 bp by sonication. Gel picture of the sonicated DNA is shown.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "DNA structure", "genetics", "epigenetics", "chromatin", "gene expression", "genomics", "Chromosome biology", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "adaptor", "ligated", "genomic"], "article_id"=>905168, "categories"=>["Biological Sciences"], "users"=>["Wei-ping Zeng", "Margaret M. McFarland"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085740.g003", "stats"=>{"downloads"=>0, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sonication_of_adaptor_ligated_genomic_DNA_/905168", "title"=>"Sonication of adaptor ligated genomic DNA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-21 02:58:10"}
• {"files"=>["https://ndownloader.figshare.com/files/1352659"], "description"=>"<p>DHS libraries or DNA in the supernatants after magnetic beads separation of naïve CD4 Tcon, nTreg cells (B–D) or primary fibroblasts (E–G) were used as templates for regular (upper panels) or real-time (lower panels) PCR analyses. Relative amplification signals in the real-time PCR were determined by comparison to the signals of DNase I-untreated total genomic DNA template amplified with primers P1 and P3 (P3 is complementary to the ligated adaptor). A. Schematic presentation of the positions of the CD4 DHS site, PCR primers and the transcription start site (TSS) of the <i>CD4</i> gene. B, E. First set of PCR using P1 and P2 primer pair. The left panel shows the PCR result using the beads-bound DHS libraries as templates; the right panel shows the PCR results using DNA remaining in the supernatants after beads isolation as templates. C, F. Second set of PCR using P1 and P3 primer pair. D, G. Third set of PCR using primer pair of P4 and P3. Tcon lib, naïve CD4 Tcon DHS library; Treg lib, naïve nTreg DHS library; Tcon sup: naïve CD4 Tcon supernatant; Treg sup, naïve nTreg supernatant; cntl, control total genomic DNA from total CD4 T cells not treated with DNase I; fibro lib, primary fibroblast DHS library; fibro sup: primary fibroblast supernatant.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "DNA structure", "genetics", "epigenetics", "chromatin", "gene expression", "genomics", "Chromosome biology", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "cd4", "dhs"], "article_id"=>905169, "categories"=>["Biological Sciences"], "users"=>["Wei-ping Zeng", "Margaret M. McFarland"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085740.g004", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Detection_of_CD4_DHS_site_by_PCR_/905169", "title"=>"Detection of CD4 DHS site by PCR.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-21 02:58:10"}
• {"files"=>["https://ndownloader.figshare.com/files/1352661"], "description"=>"<p>DNA was purified from the smears in lane 1 and 2 in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085740#pone-0085740-g004\" target=\"_blank\">Figure 4C</a>, and cloned in a pBluescript plasmid. Recombinant clones were sequenced, and the sequences were aligned to the CD4 DHS sequence. A representative clone for each population of the naïve CD4 Tcon and nTreg cells is shown.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "DNA structure", "genetics", "epigenetics", "chromatin", "gene expression", "genomics", "Chromosome biology", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "verification", "smear"], "article_id"=>905171, "categories"=>["Biological Sciences"], "users"=>["Wei-ping Zeng", "Margaret M. McFarland"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085740.g005", "stats"=>{"downloads"=>2, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sequencing_verification_of_the_smear_DNA_/905171", "title"=>"Sequencing verification of the smear DNA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-21 02:58:10"}
• {"files"=>["https://ndownloader.figshare.com/files/1352662"], "description"=>"<p>Different numbers (x10⁵) of total CD4 T cells as indicated for each lane were used to prepare nuclei for DHS analysis. Magnetic beads-isolated DNA and control total genomic DNA (cntl) were used for the first (A) and second (B) PCR as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085740#pone-0085740-g004\" target=\"_blank\">Figure 4</a>.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "DNA structure", "genetics", "epigenetics", "chromatin", "gene expression", "genomics", "Chromosome biology", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "cells", "detecting", "dhs"], "article_id"=>905172, "categories"=>["Biological Sciences"], "users"=>["Wei-ping Zeng", "Margaret M. McFarland"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085740.g006", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Determining_the_number_of_cells_required_for_detecting_a_DHS_site_/905172", "title"=>"Determining the number of cells required for detecting a DHS site.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-21 02:58:10"}
• {"files"=>["https://ndownloader.figshare.com/files/1352663"], "description"=>"<p>The CD4 DHS site was detected by real-time PCR with the indicated primer pairs. A. DHS libraries derived from nuclei of total CD4 T cells or primary fibroblasts digested with different amounts of DNase I were used as PCR templates. B. DNA in the supernatants after library isolation with magnetic beads were used as PCR templates. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type. Insets show the amplification signals using DNase I-untreated total genomic DNA of the indicated cell types as templates.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "DNA structure", "genetics", "epigenetics", "chromatin", "gene expression", "genomics", "Chromosome biology", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "cd4", "dhs", "cells", "fibroblasts", "degrees", "dnase"], "article_id"=>905173, "categories"=>["Biological Sciences"], "users"=>["Wei-ping Zeng", "Margaret M. McFarland"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085740.g007", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Detection_of_CD4_DHS_site_in_total_CD4_T_cells_and_primary_fibroblasts_with_different_degrees_of_DNase_I_digestion_/905173", "title"=>"Detection of CD4 DHS site in total CD4 T cells and primary fibroblasts with different degrees of DNase I digestion.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-21 02:58:10"}
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• {"files"=>["https://ndownloader.figshare.com/files/1352665"], "description"=>"<p>Oligonucleotide Sequences.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "DNA structure", "genetics", "epigenetics", "chromatin", "gene expression", "genomics", "Chromosome biology", "Model organisms", "Animal models", "mouse", "Molecular cell biology"], "article_id"=>905175, "categories"=>["Biological Sciences"], "users"=>["Wei-ping Zeng", "Margaret M. McFarland"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085740.t001", "stats"=>{"downloads"=>1, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Oligonucleotide_Sequences_/905175", "title"=>"Oligonucleotide Sequences.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-01-21 02:58:10"}

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