GSM6190854
Hearts and aortic valves were harvested from CTRL, ECKO, SMKO, and DKO mice after perfusion with 5 ml of 1xPBS. The tissues were immediately fixed with 4% paraformaldehyde, then washed with 20% sucrose and embedded in OCT (SAKURA Finetek USA Inc., #4583) to make frozen tissue samples. Cross-sections of the aortic valves (20 μm thickness) were mounted onto PEN-Membrane slides (Leica, #11600288) and stored at -80ºC until the LMD process. The aortic valve sections on the slide were replaced at room temperature and dried up with a handy circulator. The sections were fixed briefly with 100% ethanol and stained with hematoxylin solution (Muto Pure Chemicals Co., LTD, #30141) for 3 min, then incubated in a water bath at 30ºC for 15 min. After that, the sections were cleaned with 100% ethanol and dried up completely at room temperature. Whole aortic valve leaflets were captured using LMD7 (Leica) under the 10x magnification and collected into MaxyClear (low adhesion) microtube (Corning, PCR-02-L-C) with 20 µL 99.5% ethanol and stored at -80ºC until cDNA library preparation. cDNA library was prepared from LMD-captured aortic valves by the Nextera XT DNA library prep kit (illumina, FC-131-1024) as previously described. The amplified 0.25 ng cDNA was used for RNA-seq library preparation and sequenced on the MiSeq (illumina) with paired-end 75-base read option..