title: GSM8746288: Hanafy CMSRU | 024 | GFAP+; Mus musculus; OTHER
library_construction_protocol: Frozen sections of 4%PFA perfused prefixed mouse brains were sent to the University of Minnesota Genomics Center for probe hybridization, immunofluorescence staining, and selection of ROIs for analysis. 1x Tris-EDTA (pH9.0) solution and steaming the solution to 95⁰C method were used for antigen retrieval followed by Proteinase K digestion at 37⁰C for 5 minutes (1 µg/mL) to expose RNA targets. Library preparation was performed according to the NanoString GeoMx-NGS Readout Library Prep manual. The DSP aspirate was dried and resuspended in 10 μL DEPC-treated water, and 4 μL was used in a PCR reaction. NanoString SeqCode primers were used to amplify the tags and add Illumina adaptor sequences and sample demultiplexing barcodes. PCR products were pooled either in equal volumes or in proportion relative to AOI size, depending on the experiment, and purified with two rounds of AMPure XP beads (Beckman Coulter).Libraries were sequenced on an Illumina NextSeq 2000, according to the manufacturer's instructions, with at least 27 × 27 paired end reads.