Journal: Cold Spring Harbor protocols
Article Title: DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells
Figure Lengend Snippet: Flow chart of DNase-seq protocol. Briefly, cells are lysed with detergent to release nuclei, and the nuclei are digested with optimal concentrations of DNase I. DNase I digested DNA is embedded in low-melt gel agarose plugs to reduce additional random shearing. DNA (while still in the plugs) are then blunt-ended, extracted and ligated to biotinylated linker 1 (represented by red bars in the figure). Excess linker is removed by gel purification, and biotinylated fragments (Linker 1 plus 20 bases of genomic DNA) are digested with MmeI, and captured by streptavidin-coated Dynal beads (represented by brown balls). Linker 2 (represented by the blue bars) is ligated to the 2 base overhang generated by MmeI, and the ditagged 20 bp DNAs are amplified by PCR and sequenced by Illumina/Solexa.
Article Snippet: Baker) DNA ladder (25 bp) (Invitrogen) DNA Polymerase Buffer (1X; 10 mM Tris-Cl, pH 8.0, 50 mM NaCl, 10 mM MgCl2 , 1 mM dithiothreitol) DNase I recombinant, RNase free (10 U/μL) and incubation Buffer (10X) (Roche Diagnostics) dNTP, 10 mM (Roche Diagnostics) Dynal Streptavidin beads (Invitrogen Dynal M-280) EDTA (50 mM, pH 8.0) Ethanol (70% and 100%) Ethidium bromide(10 mg/ml) Glycogen (20 mg/mL) (Roche Diagnostics) InCert low melt agarose (1%, melted in sterile 50 mM EDTA pH 8.0 and stored at 4 °C) (Lonza) LIDS Buffer (10 mM Tris-Cl, pH 8.0,1% Lauryl sulfate lithium salt (Sigma), 100 mM EDTA) MmeI (2 U/μL, New England Biolabs) NaOAc (3 M, pH 5.3) NaOH (0.15 M) NEB Buffer 2 (10X; New England Biolabs) NEB Buffer 4 (10X; New England Biolabs) Igepal CA-630 (Sigma) Phenol (Invitrogen) Phenol: chloroform: Isoamyl Alcohol (25:24:1, Invitrogen) Phosphatase alkaline, shrimp (SAP; 1 U/μL, Roche Diagnostics) Phosphate Buffered Saline without Mg2+ /Ca2+ (1X, pH 7.2, GIBCO) Phusion DNA Polymerase (2 U/μL, Finnzymes) and Phusion HF reaction Buffer (5X) RSB Buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 ) S-adenosylmethionine (SAM; 500 μM, New England Biolabs) Spin-X filter (Fisher) T4 DNA Polymerase (New England Biolabs) TBE buffer (0.5X; 40 mM Tris-Cl, pH 8.3, 45 mM Boric Acid, 1 mM EDTA) TE Buffer (1X; 10 mM Tris-Cl, pH 8.0, 1 mM EDTA) Tris-HCl buffer (1X, pH8.0) T4 Ligase (5 U/μL) and ligation buffer (10X) (Roche Diagnostics) Trypan blue (Gibco) Ultrapure™ Agarose (Invitrogen) Ultrapure™ L.M.P.
Techniques: Flow Cytometry, Gel Purification, Generated, Amplification, Polymerase Chain Reaction