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

Sample name

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

Extraction and Methylation

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.

GC-MS method

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 m/z range of 35–550 at a scan rate of 0.34 s per scan.