Palm microbeam system

Cryo-sectioning of berries of both the varieties was carried out with Leica CM 1850 cryostat. Laser micro-dissection of specific tissues (epicarp, sarcocarp and seed) was carried out with a Zeiss Palm Microbeam system (P.A.L.M. Microlaser Technologies AG, Germany) equipped with a PALMRobo (Version: V4.6.0.4) software. An Agilent 7890A GC system, coupled to an Agilent 5975C electron ionization (EI) mass selective detector (MSD) was used for GC-MS analysis of whole extracts and laser dissected tissues. For LC-MS analysis, a UPLC-QTOF MS system (ACQUITY UPLC-Quattro Premier XE MS, Waters Corp., Milford, MA) was used. Details of the sample preparation, microscopy, and instrument parameters for mass spectrometry analysis are provided in Supplementary File S35.

A PALM MicroBeam system (Carl Zeiss Micro Imaging, Inc. Thornwood, NY, USA) laser capture microscope was used for cell dissection [61 (link), 62 (link)]. All reagents used for LCM and RNA purification were prepared in RNase-free water. Cryosectioned human tooth organs were mounted on polyethylene naphthalate (PEN) foil glass slides. After rapid hematoxylin-eosin (H&E) staining, slides were air-dried, and dental epithelial cells were identified by location and morphology. PABs and SABs were separately dissected and catapulted to Adhesive Cap (Carl Zeiss MicroImaging Gmbh, München, Germany) containing ß-Mercaptoethanol-supplemented RLT buffer (RNeasy Plus Micro Kit, RNeasy Lysis Buffer, Qiagen, Valencia, CA, USA). The caps containing buffer and cells were incubated upside-down at room temperature (RT) for 30 min and then briefly centrifuged to collect the buffer and cell complexes. RNA purification was performed with an RNeasy Plus Micro Kit (Qiagen), and reverse transcription was performed with SuperScript™ III First-Strand Synthesis System (Invitrogen Corporation, Carlsbad, CA, USA) following the manufacturer’s instructions.

Prior to LCM experiments, a cap of PCR tube was prepopulated with a 5 µL water droplet. Laser capture microdissection (LCM) was performed on a PALM MicroBeam system (Carl Zeiss MicroImaging, Munich, Germany). Voxelation of the tissue section was achieved by selecting the area on the tissue using PalmRobo software, followed by tissue cutting and catapulting. Mouse uterine tissues containing two distinct cell types (luminal epithelium and stroma) were cut at an energy level of 42 and with an iteration cycle of 2 to completely separate 100 µm × 100 µm tissue voxels at a thickness of 10 µm. The “CenterRoboLPC” function with an energy level of delta 10 and a focus level of delta 5 was used to catapult tissue voxels into the cap. The “CapCheck” function was activated to confirm successful sample collection from tissue sections to water droplets. After tissue collection into the droplet of the cap, the PCR tube was immediately centrifuged at 1000 g for 10 min at 4 °C to keep collected tissues at the bottom of the tube to avoid potential sample loss. The collected samples were processed directly or stored at −80 °C until use.