GSM8244667: RNA temporal neocortex, epileptogenic,replicate4; Homo sapiens; RNA-Seq
Each brain tissue sample was cut into small pieces and then transferred to a Dounce homogenizer with 400 μl of lysis buffer (pH 7.4, 10 mM Tris-HCL, 3 mM MgCl2, 10 mM NaCl, 0.1% IGEPAL, 0.1% Tween 20, 2% BSA). To release the nuclei, the tissues were homogenized. After 10 minutes of centrifugation at 4°C, the nuclei were washed in 1 ml of cold nuclei wash and resuspension buffer (1× PBS supplemented with 0.2 U/μl RNase inhibitor and 1% BSA). Trypan blue and Countess II FL Automated Cell Counter/Microscopy were used to evaluate lysis efficiency and viability. A 40 μm filter was used for the elimination of large clumps and cell debris. Another centrifugation step was performed, and the nuclei were collected for direct myelin removal. Then, 500 μl or an appropriate volume of nucleus wash and resuspension buffer was added to the nucleus pellet to reach the desired 700-1,200 nuclei/μl concentration. We loaded the nucleus suspension into a Chromium microfluidic chip with 3' (v2 or v3) chemical properties and labelled it with barcodes using a 10xChromium controller (10xGenomics). Following reverse transcription of RNA from the cells labelled with barcodes, sequencing libraries were prepared using reagents from a Chromium Single Cell 3' v2 or v3 reagent kit (10xGenomics) following the manufacturer's guidelines.