GSM5850306: spinal cord WT rep5; Mus musculus; OTHER
Frozen samples were embedded in OCT (TissueTek Sakura) and stored at −80 °C until cryosectioning.For cryo-  sectioning, samples were equilibrated to −22 °C and were cryosectioned at 10 μm thickness using Leica CM3050 S  Cryostat .    Sections were placed on chilled Visium Tissue Optimization Slides (3000394, 10X Genomics) and Visium Spatial Gene Expression Slides (2000233, 10X Genomics), and adhered by warming the back of the slide.     Tissue sections were then fixed in chilled methanol and stained according to the Visium Spatial Gene Expression User Guide (CG000239 Rev D, 10X Genomics) or Visium Spatial Tissue Optimization User Guide (CG000238 Rev C, 10X Genomics).     For gene expression samples, tissue was permeabilized for 24 minutes, which was selected as the optimal time based on tissue optimization time course experiments.     Brightfield histology images were taken using a 10X objective on a  Slide Scanner（Leica SCN400）.     Raw images were stitched together using Image J and exported as .tiff files with low and high resolution settings.     For tissue optimization experiments, fluorescent images were taken with a TRITC filter (ex/em brand) using a 10X objective and 200 ms exposure time. Libraries were prepared according to the Visium Spatial Gene Expression User Guide (CG000239 Rev D, 10X Genomics).    Libraries were loaded at 300 pM and sequenced on a NovaSeq 6000 System (Illumina) using a NovaSeq S4 Reagent Kit (200 cycles, 20027466, Illumina. Sequencing was performed using the following read protocol: read 1, 28 cycles; i7 index read, 10 cycles; i5 index read, 10 cycles; read 2, 90 cycles.