title: GSM4672357: ATAC-seq in human aortic valve interstitial cells; Homo sapiens; ATAC-seq
library_construction_protocol: 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. Nuclei from approximatively 2.5-4 x 105 cells were prepared as previously described (Shashikant T, et al. 2018, BMC Genomics). Libraries were first amplified for 5 cycles using custom-synthesized index primers and then a second amplification was performed. The appropriate number of additional PCR cycles was determined using quantitative real-time PCR by plotting the Rn value (fluorescent signal from SYBR Green I) versus cycle number and determining the cycle number corresponding to one-third of the maximum fluorescent intensity. Libraries were PCR-amplified for 4-7 cycles. After PCR amplification, libraries were purified using DNA clean up & concentrator kit (Zymo Research, Cedarlane, Canada)Library quality was assessed on a BioAnalyser (Agilent, ON, Canada) using Agilent High Sensitivity DNA kit (Agilent, ON, Canada). Sequencing was performed on an Illumina HiSeq4000 (UCSD IGM Genomics Facility, CA, USA).
identifiers.namespace: GEO
identifiers.id: GSM4672357