title: GSM9002100: MCF7_MED1WT_PolII_CnR; Homo sapiens; OTHER
library_name: GSM9002100
library_construction_protocol: Trypsin-digested cells (5×10^5 cells) were harvested and fixed with freshly prepared 0.5% PFA for 2 min. After stopping the cross-linking using 500 μl 2.5 M Glycine, cells were washed three times with digitonin wash buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM spermidine, protein inhibitor cocktail, 0.005% digitonin, 0.05% SDS and 1% Triton X100) and bound to Concanavalin A coated beads (Polysciences, Cat# 86057-10) pre-washed by binding buffer (20 mM HEPES, pH 7.4, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). Cells on beads were then incubated with antibodies on a nutator overnight at 4 °C. After overnight incubation, cells were washed three times with 0.005% digitonin wash buffer, resuspended in 100 μl digitonin buffer containing Protein A/G-Micrococcal Nuclease fusion protein (pAG-MNase) and nutated in 4 °C for 1.5 hour. After pAG-MNase binding, cells were washed three times with digitonin wash buffer for 5 min. Finally, cells were resuspended in 100 μl ice cold digitonin buffer in a heating block pre-cooled in ice water bath. Digestion of the chromatin was initiated by addition of 2 mM CaCl2 in the tube, and lasted for 4 hours at 0 °C. The digestion was stopped by addition of 100 μl 2x STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 100 μg/ml RNase A, Glycogen 50 μg/ml). Digested DNA was released by incubation at 37 °C for 30 min, and supernatant was collected. Then 5 μl proteinase K (20 mg/ml) and 200 μl 2x elution buffer (20 mM Tris-HCl (pH=8.0), 300 mM NaCl, 20 mM EDTA, 2% SDS, 10 mM DTT) was added to each sample for reverse crosslinking at 65 °C overnight. DNA was extracted by phenol-chloroform and dissolved in low EDTA TE buffer. Libraries were generated using DNA SMART kit or ThruPLEX kit (both from Takara Bio) according to the manufacturer's instructions. After size selection (for 250-600bp) by AMPure XP beads (Beckman Coulter), the library was quality was evaluated using the Bioanalyzer and sequenced on Illumina NovaSeq SP (100bp two ends) and Illumina NextSeq High (75 bp single end).
identifiers.namespace: GEO
identifiers.id: GSM9002100_r1