GSM6449903: ID216_DNaseI; Homo sapiens; OTHER
DNase I digestions of permeabilized cells were performed briefly as follows: live cells were added directly to a solution of DNase I (DPFF, Worthington) in dilute Nonidet P40, digested for 3 min at 22oC, and the reactions then terminated by addition of SDS to 0.5%. DNase I was typically used in the range of 2-6 μg/ml using a final 1.5 x 107 cells/ml.  DNase I (DPFF) was obtained from Worthington Biochemical Corporation and typically used in the range of 2-6 μg/ml using a final 1.5 x 107 cells/ml. The DNA digestion extent was comparable in all the generated samples as measured by RT-PCR. DNase-Seq libraries were then prepared. Levels of DNase I digestion were assessed using quantitative real-time PCR, measuring the ratio of the presence of known DNase I hypersensitive regions compared to a more resistant inactive region. Sequences of the PCR primers used for this purpose were, for the active region, TBP promoter 5´- CTGGCGGAAGTGACATTATCAA and 5´- GCCAGCGGAAGCGAAGTTA; and for the inactive region, a region of chromosome 18: 5´- ACTCCCCTTTCATGCTTCTG and 5´- AGGTCCCAGGACATATCCATT. DNase-Seq samples were generated from a size selection of DNase I-digested DNA fragments comprised within a range of 100 to 250 bp (not including linkers) and subjected to library preparation as per manufacturer´s instruction (Illumina).