GSM8635801: Control siRNA Replicate 1; Mus musculus; ATAC-seq
3T3-L1 cells were cultured and treated as described above in 15 cm diameter plates.  The cells were transfected with siRNAs targeting Parp7 or a universal control 24hrs before the addition of the differentiation cocktail. The cells were then differentiated for 24hrs Cells were harvested by trypsinization and counted using the BioRad TC20 automated cell counter. 50,000 cells were collected and pelleted by centrifugation. Samples were then resuspended in buffer containing 10mM Tris-HCL pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP-40, 0.1% Tween-20, and 0.01% digitonin, and incubated on ice for 3 min. Nuclei were then washed twice with buffer containing 10mM Tris-HCL pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20. Nuclei were pelleted by spinning at 500g for 10 min at 4°C in a fixed angle centrifuge. Pellets were then resuspended in transposition mixture containing 20mM Tris-HCL pH 7.6, 10mM MgCl2, 20% DMF, 0.01% Digitonin, 0.1% Tween-20, and 100 nM recombinant Tn5 transposase. After pipetting up and down 6 times, mixture was incubated at 37°C for 30 mins with 1000 RPM mixing using the Eppendorf Thermomixer C. Resulting tagmented DNA was purified using Qiagen PCR CleanUp columns and was eluted with elution buffer. Samples were then combined with 2X NEBNext High-Fidelity Master Mix (NEB cat#M0541L), Nextera i7 and i5 indexed primers, and amplified by PCR: 1) 72C 5Min, 2) 98°C 30Sec, 3) 98°C 10Sec, 4) 63°C 10Sec. PCR steps 3-4 were repeated 8 times. Adaptor contamination was removed from libraries using 1.3X volume AMPure beads and eluted in 10mM Tris-HCL (pH 8). n= 2 biological replicates