title: GSM7111984: 6-postATN Spatial Transcriptomics; Mus musculus; OTHER
library_construction_protocol: scRNA-seq: Microdissect, dissociate, and pool cells across acute brain slices in B6 mice; spatial transcriptomics: flash frozen brains, cryosectioning 10um and place on 10xgenomics visium slides, tissue staining for NeuN and DAPI, permeabilization and library construction following manufacturer's protocol For scRNA-seq: Cells were placed into 8-well strips containing cell collection buffer, and each strip of cells was flash frozen on dry ice, then stored at -80°C until cDNA synthesis. For cDNA synthesis, cells were lysed by adding 1 µL lysis mix and 1 µL 10 µM barcoded RT primer. The samples were incubated for 5 minutes at 50°C to lyse the cells, followed by 20 minutes at 80°C to inactivate the Proteinase K. Reverse transcription master mix was added to the lysis reaction and incubated at 42°C for 1.5 hour, followed by 10 minutes at 75°C to inactivate reverse transcriptase. PCR was performed, and samples were pooled across the plate to yield approximately 2 mL pooled PCR reaction. From this, 500 µL was purified, washed twice with 75% ethanol, and eluted in 20 µL nuclease-free water. For Visium Spatial transcriptomics: as described by 10x genomics for Visium workflow with 24min permeabilization time