ONLINE METHODS Sample Preparation for Lipid Standards

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Lipid standards were purchased from Avanti Polar Lipids, Inc., and Matreya LLC and a list is included in the supplementary information section. A final concentration of 1 µM was desired for the LC-IMS-MS studies. The standards were diluted with chloroform to reach the desired concentration. River Laboratory (Raleigh, NC). Females were mated with fertile males of the same strain to induce pregnancy. Mice were euthanized between 0900 and 1000 h on day 8 of pregnancy (0 h on day 1 = vaginal plug). Day 8 implantation sites were microdissected to isolate the top and bottom hemispheres for subsequent lipid extraction. Due to the high sensitivity of our mass spectrometry platforms each dataset represents either a top or bottom hemisphere from a single implantation site. In total 12 samples were analyzed; top and bottom hemispheres from 6 implantation sites collected from two mice (3 implantation sites from each mouse). Lipids were extracted from the tissue using a Folch extraction [1]. Briefly, tissue was lysed in 400 µl methanol using a tissue lyser with a 3 mm tungsten carbide bead for 3 min. Sample was transferred into a vial and 800 µl of chloroform was added. The sample was vortexed for 60 sec then shaken for 1hr at room temperature at 1000 rpm. The samples were then vortexed briefly and 300 µl of water was added to induce a bi-phase separation. The sample was gently mixed, incubated at room temperature for 10 min and then centrifuged for 5 min at 15,000 x g at 4C. The lower organic layer was collected and transferred into another vial. The remaining Electronic Supplementary Material (ESI) for Analyst.

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تاریخ انتشار 2015