GSM7995348: HUDEP-2 WT DNase-I 60U/mL; Homo sapiens; OTHER
Nuclei from 100,000-200,000 cells were extracted in the presence of 0.04% de-ionized IGEPAL CA-630 incubated at 4 ºC for 10 min. Nuclei were treated with a gradient of DNase I solution (40 IU to 100 IU of DNase I) for 3 min at 37 ºC. DNase I digestion was quenched by adding an equal volume of 5X Stop buffer and 20μL RNase A (Sigma, R4642) followed by incubation at 37 ºC for 60min. After incubation, 1μL of Proteinase K (Sigma, P4850) was added and the reactions were incubated at 50 ºC for 60min. Digested genomic DNA was visualized on 1.2% agarose gen and the fragment size profile was generated using the Fragment Analyzer (Advanced Analytical). Prior to genomic library generation, fragments were subjected to size selection with large fragment depletion by magnetic bead separation. Fragment size distribution and concentration of the fractionated sample was measured with Fragment Analyzer (Advanced Analytical). Illumina compatible, double-stranded DNA library libraries from the size fractionated samples were constructed using the ThruPLEX DNA-seq Kit (Takara Bio) according to manufacturer's instructions.