GSM8969664: Late passage iPSC WT WGBS; Homo sapiens; Bisulfite-Seq
Proliferating and G2-arrested iPSCs were harvested using a single-cell suspension (see growth protocol) and snap-frozen for further use. A subset of G2-arrested cells was taken for cell cycle analysis to verify the arrest. Fort the cell cycle analysis cells were collected using a single-cell split and quenched with DPBS. 1M cells were transferred into a protein lo-bind Eppendorf tube (#) and centrifuged. The supernatant was discarded and the cell pellet resuspended in 100ul PBS-T (0.2% Tween 20, Thermo Fisher Scientific, #J20605.AP) To permeabilize and fix the cells 1ml of 70% EtOH was added dropwise for a final concentration 1ml/1M cells and incubated at 4°C for two hours or overnight. For one wash the cell suspension was then centrifuged and resuspended in 1ml PBS-T. After centrifuging the cell pellet was in 1ml Dapi solution of 0.1% Triton-PBS (Triton-X-100. Thermo Fisher Scientific, #85111) and 1ug/ml Dapi (Invitrogen, #D1306) and incubated at RT for 30 minutes in the dark. The cells were washed with 1ml PBS-T. Finally, the cells were resuspended in 200ul DPBS-10% FBS and filtered for FACS analysis. Endoderm differentiated cells were harvested using a single-cell suspension (see growth protocol) and prepared for FACS analysis. ZIP8K8 single-cell suspensions were quenched DPBS-10% Fetal bovine serum (FBS, PAN Biotech, #P30-2602)). Next, cells were centrifuged at 4°C. Cells were then resuspended in DPBS-10% FBS containing the surface marker anti-CXCR4 antibody (Biologend, #306506) in a 1:200 dilution and incubated for 15 min at 4 °C in the dark. Next, cells were washed once again with DPBS-10% FBS and centrifuged at 4°C before FACS. Finally, cells were resuspended in DPBS-10% FBS and passed through a 70µm cell strainer (Corning, #431751). NCAM positive cells were sorted and snap-frozen for further use. FACS analysis was performed on the FACSCelesta Flow Cytometer (Beckton Dickinson). BiSulfite-seq