title: GSM6528223: Bulk RNA-seq E3_Young_Bulk RNA-seq rep1; Mus musculus; RNA-Seq
library_construction_protocol: The mirroring hemisphere (right) from brains processed for scRNAseq were immediately placed in OCT compound (Fisher HealthCare Tissue Plus O.C.T. Compound Clear 4585) and gently lowered into isopentane (Sigma Aldrich 2-Methylbutane M32631) in a beaker surrounded by dry ice (isopentane chilled to approximately -70°C). Brains were submerged for 60 seconds, placed on dry ice, wrapped in aluminum foil, and stored at -80°C until sectioning. Prepared brain hemispheres were cryosectioned to 10 μm thick coronal sections at approximately Bregma -2.00 mm. Serial 10 μm sections immediately rostral and caudal to the section mounted on the Visium Spatial Gene Expression slide (10X Genomics) were collected for immunohistochemistry. Optimal tissue permeabilization time was determined using the manufacturer's optimization protocols (10X Genomics, Visium Spatial Tissue Optimization), and accordingly, experimental tissues were permeabilized for 18 min for Visium Spatial Gene Expression analysis. Prior to library preparation, tissue sections were methanol-fixed, stained with hematoxylin and eosin (H&E), and imaged on a Nikon NiU microscope with Fi3 color camera. Sections were then permeabilized and processed to obtain cDNA libraries, which were subsequently prepared according to the manufacturer's protocol (https://support.10xgenomics.com/spatial-gene-expression/libraryprep). Sample libraries were constructed using Next GEM automated 3' reagents (10X Genomics, v3.1) following manufacturer's suggested protocol (#CG000286 Rev B). Final library quantification and quality check was performed using BioAnalyzer (Agilent), and sequencing performed on a NovaSeq 6000 S4 flow cell, 150 bp Paired-End sequencing (Novogene).