GSM6504779: ASAP16 Ctl PT231 PFCTX 5'; Homo sapiens; RNA-Seq
Postmortem brain tissue from controls with no known history of neurological or neuropsychiatric symptoms was acquired from the Cambridge Brain Bank (ethical approval from the London-Bloomsbury Research Ethics Committee, REC reference:16/LO/0508). Brains were bisected in the sagittal plane with one half flash-frozen and stored at -80 °C and the other half fixed in 10% neutral buffered formalin for 2–3 weeks. From the flash-frozen blocks, 50-100mg were sampled from the dorsolateral prefrontal cortex (Brodmann area 46) and stored at -80 °C until use.  Nuclei was isolated as previously described with minor modifications42. Around 20 μg of -80 C-conserved tissue were thawed and dissociated in ice-cold lysis buffer (0.32M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl pH 8.0, 1 mM DTT) using a 1 ml glass douncer (Wheaton). The homogenate was slowly and carefully layered in the centrifuge tubes on top of a sucrose layer (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl pH 8.0, 1 mM DTT) to create a gradient, and then centrifuged at 15500 rpm for 2 h 15 min. After centrifugation, the supernatant was removed, and the pellet softened for 10 minutes in 100 μl of nuclear storage buffer (15% sucrose, 10 mM Tris-HCl pH 7.2, 70 mM KCl, 2 mM MgCl2) before resuspension in 300 μl of dilution buffer (10 mM Tris-HCl pH 7.2, 70 mM KCl, 2 mM MgCl2, Draq7 1:1000). The suspension was then filtered (70 μm cell strainer) and sorted via FACS (FACS Aria III, BD Biosciences) at 4° C with low flowrate, using a 100 μm nozzle (Pipette tips and Eppendorf tubes for transferring nuclei were pre-coated with 1% BSA). 8500 nuclei were sorted for single-nuclei RNA-sequencing and then loaded onto the Chromium Next GEM Single Cell 5' Kit (10x Genomics, PN-1000263). Sequencing libraries were generated with unique dual indices (TT set A) and pooled for sequencing on a Novaseq6000 using a 100-cycle kit and 28-10-10-90 reads.