GSM7733714: iPSC WT G2 arrest 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). Genomic DNA was extracted using Phenol-chloroform purification. In brief cell pellets were resuspended in 200ul DPBS an200ul cold phenol-chloroform was added (1:1 ratio), after mixing by vortexing the cell solution was centrifuged for 10 minutes at full speed. The aqueous layer was transferred to a fresh DNA-lo bind Eppendorf tube and supplemented with 10ul of 5M NaOH, 2ul glycogen, and 500ul 100% EtOH. After gentle vortexing the solution was incubated at -20°C over night. The next day, ethanol precipitation was performed by centrifuging the cell solution at 4°C for one hour at full speed. After discarding of the supernatant, the pellet was resuspended on 1ml of cold 70% EtOH and centrifuged again at 4°C for one hour at full speed. Finally, after discarding the supernatant and drying the pellet for 10 minutes the DNA was resuspended in 25ul of low EDTA TE buffer (Swift Biosciences, #90296). In a fresh lo-bind DNA tube dilute 300ng genomic DNA in 50ul low EDTA TE buffer and transfer to a Covaris tube (#520045) for fragmentation.  Next the DNA was fragmented using the following sonication settings: 10 % cycle, PIP of 175, and 200 bursts/cycle for 2 x 45 second. The fragmented DNA was then transferred to a DNA lo-bind tube and purified using the Zymo Clean & Concentrator 5 Kit (#D4013) following the manufacturer's instructions for fragmented DNA. The final product was eluted in in 20ul low EDTA TE buffer. The purified genomic DNA was the bisulfite converted using the Zymo EZ DNA Methylation-Gold Kit (#D5005) by following the manufacturer's instructions. The final product was eluted in in 15ul low EDTA TE buffer. Finally, the sequencing library was prepared using Accel-NGS Methyl-seq DNA library kit (Swift Biosciences, #30024-SWI). The libraries were purified using Agencourt AMPure XP beads (Beckman Coulter, #A63881), and diluted to 4nM for sequencing.