GSM7990372: FCPN, Layer5, CTR, h306R_Layer5; Homo sapiens; RNA-Seq
Human brain specimens for Down Syndrome were procured in part as a gift of Dr. Jerzy Weigel at the Institute for Basic Research (Staten Island, NY) and in part from the NIH NeuroBiobank.  Non cognitively impaired (NCI) control human brain tissue was obtained from the NIH NeuroBioBank. Human brain tissue arrived frozen on dry ice, blocked for the BA9 frontal cortex region.  Tissue blocks were kept frozen to preserve tissue and RNA quality, and were cryostat cut (-25 0C) at 20 μm-thick tissue sections in the coronal plane to identify the cortical layers (CM1860UV, Leica, Buffalo Grove, IL) and mounted on polyethylene naphthalate (PEN) membrane slides (Leica).  Slides were kept under desiccant at -80 0C until used for immunohistochemistry. RNase-free precautions were employed, and solutions were made with 18.2 mega Ohm RNase-free water.  Slides were equilibrated to room temperature under dessicant to reduce condensation. Nissl (thionin) stained and air dried before proceeding directly to laser capture microscopy.  Semi-automated detection was used to identify layer III and layer V pyramidal neurons for isolation via LCM. Once cells were confirmed as pyramidal neurons by morphology, cutting was performed, dropping cells directly into lysis buffer (Qiazol).  Max LCM time was 1 hour per slide to preserve RNA quality.   RNA from ~500 L3 and L5 BA9 neurons from each brain specimen were combined and purified using miRNeasy Micro kit (Qiagen) according to manufacturers' specifications, along with two rounds of the optional DNase digestion before the final washes. Each RNA Sample was split into two technical replicates before library preparation using SMARTer Stranded Total RNA-Seq kit-Pico input Mammalian. Dual Index primers and UMIs were unique to each technical replicate.  Fragmentation was performed as needed based on RIN/DV200 values according to manufacturers' recommendation. RNA-seq samples were quantified (Agilent Tapestation, D1000 DNA Assay), and samples below 2 nM of the library were excluded. Samples were pooled in equimolar concentrations and assayed on Illumina NovaSeq 6000 S1 and S2 flow cells using the 100-cycle paired-end read protocol at the NYUGSOM Genome Technology Center.