GSM8709903: FCPN, Layer3, MCI, 325_L3; Homo sapiens; RNA-Seq
Brain tissues obtained from postmortem autopsy cases (n=20) with an antemortem clinical diagnosis of NCI, MCI, and AD from the NIH NeuroBioBank housed at the University of Miami Brain Endowment Bank (UMBEB) were examined. Frozen unfixed 20 um thick tissue sections from BA9 tissue blocks were cut on a cryostat (CM1860 UV, Leica, Buffalo Grove, IL) in the coronal plane and thaw mounted on polyethylene naphthalate (PEN) membrane slides (Leica). Slides were kept under desiccant at -80 °C until used for LCM. Stringent RNase-free precautions were employed, and solutions were made with RNase-free water (ThermoFisher, Waltham, MA). PEN membranes were equilibrated to room temperature (RT) under desiccant (-20 °C for 10 min, 4 °C for 10 min, RT for 1 min) before the Nissl stain procedure (Alldred et al., 2024). Slides were rinsed in phosphate-buffered saline (PBS, pH 7.4), and immersed in a Nissl stain solution (0.18 M sodium acetate, 0.6% acetic acid, 0.1% thionin; Sigma, St. Louis, MO) for 1 min, then rinsed with PBS twice. Slides were air-dried for 5 min and then immediately utilized for LCM of pyramidal neurons. An LCM system (LMD7000; Leica) was employed to identify and microisolate pyramidal neurons using a 40x objective (PL-Fluotar NA 0.60). Neurons from layer III were outlined using the draw/cut tool (Leica LMD version 8.2.1) and collected by gravity into 50 ul of Qiazol extraction solution (Qiagen, Germantown, MD) (Fig. 1 A-C) (Alldred et al., 2024). Captured cells were counted and frozen at -80 °C until 600-800 cells per region were isolated. An average of 9 tissue sections per subject were required to collect sufficient numbers of Nissl-stained layer III pyramidal neurons.RNA from 600-800 pyramidal neurons was purified using miRNase Micro kit (Qiagen) according to manufacturers' specifications with the DNase digest performed twice sequentially (Alldred et al., 2024). RNA quality control was assessed (RNA 6000 pico kit, Agilent, Santa Clara, CA). The SMARTer Stranded Total RNA-Seq Kit v3 Pico Input Mammalian (Takara Bio, Mountain View, CA) was employed with 8 ul of input RNA. Samples were quantified (2100 HS DNA kit; Agilent) with a minimum of 2 nM required for downstream applications (Alldred et al., 2024). Samples were pooled in equimolar concentrations and assayed on the NovaSeq 6000 using the S1 100 Cycle Flow Cell v1.5 (Illumina, San Diego, CA) at the NYU Grossman School of Medicine Genome Technology Center (NYUGSOM GTC).