GSM7498575: CD34, GATA1, rep1; Homo sapiens; OTHER
5x10E5 cells were harvested from CD34+ HSPCs expanded for 48 hours post-thawing and bound to ConA-coating beads by rotation for 10 minutes at room temperature. Cells were permeabilized (20 mM HEPES pH 7.5, 150 mM NaCL, 0.5 mM Sperimidine, Roche Complete EDTA free 5892953001, 0.05% w/v digitonin) and incubated overnight at 4°C with anti-CUX1 (PUC) 1:50, anti-SMARCA4 (Cell Signaling, 49360s) 1:100, or anti-GATA1 (Abcam, ab181544) 1:100 antibodies. Protein A/G-MNase beads were added and placed on a tube rotator for 1 hour at 4°C. MNAse bound DNA was cleaved and released by adding 1X pA-MNAse mix containing CaCl2 at 0°C for 30 minutes, STOP buffer was added and CUT&RUN fragments were released by incubating for 30 minutes at 37°C. Library end repair, ligation, and amplification were performed using the Ovation Ultralow System V2 kit (Tecan Genomics Inc. 0344NB-32) and amplified by PCR with the following parameters: 1 cycle of 72°C  2 minutes, 95°C  3 minutes, followed by 13 cycles of 98°C  20 seconds, 65°C  30 seconds, 72°C  30 seconds, and a final extension at 72°C  for 1 min.  Libraries were cleaned up using MinElute PCR purification kit (Qiagen) and a left-sided size selection using SPRI beads (Beckman Coulter B23317). Final libraries were analyzed by Bioanalyzer (Agilent) prior to sequencing.