GC-MS raw data_Figure 6E_Lysophosphatidic Acid Shifts Metabolic and Transcriptional Landscapes to Induce a Distinct Cellular State in Human Pluripotent Stem Cells

<strong>Sample name</strong> hESCs (H1 cells) were given treatments for two days and then collected for GC-MS analysis. E8: E8 medium AX: E8 + 1.6% AlbuMAX; BSA: E8 + 1% albumin; BSA+hCDL: E8 + 1% albumin + 0.1% hCDL; LPA+BSA: E8 + 1 μM LPA + 1% albumin; LPA+BSA+hCDL: E8 + 1 μM LPA + 1% albumin + 0.1% hCDL STD: standard lipids mixture used as reference <strong>Extraction and Methylation</strong> Sample preparation was conducted according to the previously reported method (Araujo et al., 2008) with the modification. Briefly, spent medium was removed, and cells were rinsed with 1 mL/well 0.9% (w/v) saline twice. Then 0.5 mL/well -80°C 80% methanol was added to quench the metabolism. Five wells of cells (from 6-well plate) were scrapped off into a glass screw-cap tube. Then 4 mL heptadecanoate containing chloroform (4 μg/mL, internal standard for fatty acids) was added into the tube. Vortex, and then centrifuge at 2000 rpm for 5 min. Cellular debris was carefully removed, and nitrogen blow the solution till dry. Add 1.5 mL hexane and 1.5 mL 14% boron trifluoride (BF₃)/methanol solution. Seal the tube with nitrogen gas, heat it at 100°C for 1 h using MK200-2 dry bath incubator (Aosheng), and then cool down to room temperature. Add 1 mL water into the tube, vortex and then centrifuge at 3000 rpm for 10 min. The upper layer was transferred into a new 1.5-mL eppendorf tube and evaporated by nitrogen gas. The residue was re-dissolved in 100 μL hexane for GC-MS analysis. <strong>GC-MS method</strong> Samples were analyzed using an Agilent GC-MS system (Agilent) consisting of a 6890 gas chromatography and a 5973 mass spectrometer. Fatty acid methyl esters were separated by an Omegawax™ 250 fused silica capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness, Supelco, Bellefonte, PA). The optimized oven temperature program was: initial temperature set at 180°C and held for 3 min; ramped to 206°C at 2°C/min and held at 206°C for 25 min, then, ramped to 240°C at 10°C/min and held for 5 min. Overall, the total run time was 50 min. Carrier gas was high-purity helium at a flow rate of 1.5 mL/min. Injector temperature was set at 250°C. Injection volume was 2 μL with a split ratio of 1:15. The mass spectrometer was operated in electron-impact (EI) mode at 70 eV ionization energy. The temperatures of quadrupole and ionization source were set at 150°C and 280°C, respectively. The spectra from 3 to 50 min were acquired with the <em>m/z</em> range of 35–550 at a scan rate of 0.34 s per scan.