Assay summary for DNAse-seq_Zon_Lab_0002AS

Assay Type:
Zon Lab, Boston Children's Hospital
Zebrafish peripheral blood was collected by cardiac puncture in anesthetized adult zebrafish. As the mature erythrocytes in lower vertebrates retain their nuclei, we are able to isolate erythrocyte nuclei from peripheral blood. DNase I hypersensitivity assays were arried out. Briefly, red blood cells were treated with 0.025% Igepal in Buffer A (15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM Spermidine) for 8 minutes. The released nuclei were pelleted and incubated for 3 minutes at 37C in DNase buffer (13.5mM Tris-Cl pH8.0, 88.5 mM NaCl, 54 mM KCl, 0.9 mM EDTA, 0.45 mM EGTA, 0.45 mM Spermidine, 6 mM CaCl2) plus 0, 40, 60, 80 or120 units/ml of DNaseI (Sigma Aldrich). The reaction was stopped by the addition of an equivalent volume of Stop Buffer (50mM Tris-Cl pH 8.0, 100 mM NaCl, 0.10 %SDS, 100 mM EDTA) and incubated for 1 hr with 1100 units/ml Proteinase K. Small DNase I fragments from the 120 unit DNaseI treatement were enriched using a sucrose gradient. These ends were then labeled and amplified for Solexa sequencing Small genomic DNA fragments resulted from DNase I treatment of intact nuclei were enriched using a sucrose gradient centrifugation after optimal DNase I treatment of isolated nuclei.
Added by User:
Date Added:
June 2, 2016, 3:02 p.m.
Last Edited:
July 25, 2018, 5:47 a.m.