GSM4672364: H3K27ac HiChIP in human aortic valve interstitial cells; Homo sapiens; Hi-C
Human aortic valve interstitial cells were isolated from control non-mineralized aortic valves obtained from patients undergoing heart transplantation. The protocol was approved by the local ethical committee and informed consent was obtained from the subjects. Aortic leaflets were cut into pieces and incubated in 0.3% type I collagenase (Invitrogen, Thermo Fisher Scientific, ON, Canada) at 37°C for 45 minutes, then filtered through a 70 µm mesh, centrifuged for 5 minutes at 1,500 rpm and resuspended in complete media (DMEM, 10% FBS with L-glutamine and sodium pyruvate). Cells were used between passages 3 to 7. H3K27ac HiChIP was performed from about 1 million of cells as described previously (Mumbach et al., 2016, Nat Methods). Library was generated in 50 µL of a PCR master mix (25 µL of Phusion HF 2X (New England BioLabs, ON, Canada), 1 µL of each Nextera Ad1_noMX and Nextera Ad2.X at 12.5 µM, and 23 µL of water). The following PCR program was performed: 72 °C for 5 min, 98 °C for 1 min, then cycle at 98 °C for 15 s, 63 °C for 30 s, and 72 °C for 1 min. Cycle number was set to 10 considering the amount of DNA. Library was placed on a magnet and eluted into a new tube before purification with clean up & concentrator kit (Zymo Research, Cedarlane, Canada). Library size selection (300-700 bp) was performed by cutting a 2% agarose gel slice and DNA purified with PureLink Quick Gel Extraction (Thermo Fisher, ON, Canada). Library quality was assessed on a BioAnalyser (Agilent, ON, Canada) using Agilent High Sensitivity DNA kit (Agilent, ON, Canada). Paired-end sequencing with 500 million reads was performed with NovaSeq 6000 S4 (UC San Diego IGM Genomics Center). HiChIP
GEO
GSM4672364
GEO Accession
GSM4672364