title: GSM2579633: HCT116.25000.NicE-seq; Homo sapiens; OTHER
library_construction_protocol: For NicE-seq and ATAC-seq, genomic DNA was extracted using phenol chloroform method. For NicE-seq, varying number of crosslinked and native cells were used. Nuclei were isolated by incubating the cross-linked cells in cytosolic buffer (15 mM Tris-HCl pH 7.5, 5 mM MgCl2, 60 mM KCl, 0.5 mM DTT, 15 mM NaCl, 300 mM sucrose and 1% NP40) for 10 min on ice with occasional agitation. Nuclei were precipitated by spinning at 1000 × g, 4 °C for 5 min and the supernatant was discarded. Open chromatin DNA was labeled with biotin by incubating the nuclei in presence of 2.5 U of Nt.CviPII (NEB R0626S), 50 U of DNA polymerase I (M0209S) and 30 μM of each dNTP including 6 μM of biotin-14-dATP (Invitrogen, 19524-016) and 6 μM of biotin-16-dCTP (ChemCyte, CC-6003-1) in 200 μL of 1 × NEB buffer 2. Labeling reaction was carried out at 37°C in a thermo-mixer at 800 RPM for 2 h. 20 μL of 0.5 M EDTA and 2 μg of RNase A was added to the labeling reaction and incubated at 37°C for 0.5 h to stop the labeling reaction and digest RNA. Finally DNA-protein cross-linking was reversed by adding 20 μL of proteinase K (NEB, P8107S) and 20 μL of 20% SDS to the reaction and incubating at 65°C for 1 h. Biotin labeled genomic DNA was extracted using phenol chloroform. Open chromatin labeling in native cells was performed without formaldehyde crosslinking step. Biotin-labeled genomic DNA from fixed (HCT116, MCF7 and decitabine treated HCT116 cells) and native (HCT116) reactions were sonicated into 150 bp fragments (Covaris) and 1 μg of DNA was end-repaired, dA-tailed and ligated with NEB Illumina adaptor (NEB, E7370S). Without further purification, the ligation product was mixed with 50 μL of Streptavidin magnetic beads (Invitrogen 65001, blocked using 0.1% cold fish gelatin in 1 × PBS overnight at 4°C) in 1 mL of B&W buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 M NaCl). Biotin-labeled open chromatin DNA was captured by streptavidin at 4°C for 2 h with end-over-end rotation. The beads were washed four times with B&W buffer plus 0.005% of Triton X-100 followed by one wash with TE plus Triton X-100. The beads were resuspended in 40 μL of nuclease free water and 4 μL was used for library amplification using PCR (NEB, #E7370S). 10 PCR cycles were usually sufficient to generate the amount of library DNA needed for sequencing.  For library construction using 250 cells, all procedures remained same except addition of 10 μg of glycogen during genomic DNA extraction. In the case of 25 cells, 0.25 U of Nt.CviPII and 5 U of DNA polymerase I were used. The entire DNA was sonicated, 10 μL of streptavidin beads were used to capture the biotinylated DNA and were used as template for library amplification. For input library preparation 1 µg of purified genomic DNA was used instead of the labeled chromatin.
attributes.tag: GEO Accession
attributes.value: GSM2579633