GSM7732669: D578_ITD-15_FLT3i; Homo sapiens; OTHER
DNaseI-seq was performed on purified CD34+ or CD117+ blasts from fresh patient samples. Live cells were added directly to a solution of DNase I in dilute NP40, digested for 3 min at 22˚C, and the reactions then terminated by addition of SDS to 0.5%. DNase I (Worthington Biochemical Corporation) was typically used in the range of 2-6 μg/ml using a final 1.5 x 107 cells/ml. Cell lysates were treated with RNAse A for 1 h at 37 ˚C and DNA was isolated by phenol/chloroform extraction. DNaseI-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 libraries were made using a Kapa Hyper prep kit (Roche) according to manufacturers instructions. 12-14 cycles of PCR amplification were used and 200-450 bp fragments were size selected by gel electrophoresis o na 1.5% agarose gel. Libraries were validated by qPCR and quantified using the High Sensitivity DNA kit (Agilent) and Kapa Library Quantification kit (Roche) prior to sequencing on a Nextseq 2000 75 using a NextSeq 500/550 High output kit.