GSM8759581: 22Rv1,Ctrl, Nucleolar DNA; Homo sapiens; OTHER
Cells were cross-linked by 1% formaldehyde and quenched with 125 mM glycine upon reaching 80% confluence. Then they were resuspended in 2 ml of buffer A (10 mM HEPES, pH 7.9; 10 mM KCl; 1.5 mM MgCl2; 0.5 mM DTT) and incubated on ice for 5 min. The cell suspension was transferred to a precooled 2-ml Dounce tissue homogenizer and homogenized 10 times. Subsequently, the homogenized cells were centrifuged and rough nuclei were contained in the pellets. The pellets were resuspended with 3 ml S1 solution (0.25 M sucrose; 10 mM MgCl2), and the liquid was then carefully layered over 3 ml S2 solution (0.35 M sucrose; 0.5 mM MgCl2) ensuring that the two layers remained distinctly separated. The mixture was centrifuged at 2500 rpm for 5 min at 4 °C to obtain a cleaner nuclear pellet. The pellets were resuspended with 3 ml S2 solution and subjected to sonication with six 10-s bursts to disrupt nuclear membranes. The liquid was then layered over 3 ml S3 solution (0.88 M sucrose; 0.5 mM MgCl2), and then centrifuged at 3500 rpm for 10 min at 4 °C. The supernatant was discarded, and the pure isolated nucleoli were collected. Total genomic and nucleolar DNA was isolated with TIANamp Genomic DNA Kit (TIANGEN, DP104) and dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.9). After shearing the DNA by sonication, libraries were constructed using ThruPLEX DNA-seq kit (Rubicon Genomics).