GSM5841552: Advanced 1; Mus musculus; RNA-Seq
Evan's blue dye was administered intradermal into the mouse hindpaw to identify the PLNs. The PLNs were harvested, embedded in Optimal Cutting Temperature (OCT) compound (Cat#4583; Sakura, Osaka, Japan), frozen in liquid nitrogen, and stored at -80C. The lymph nodes were patterned to identify PLNs from individual limbs to relate with arthritic severity. A single 10um tissue section was placed on each capture area of a Gene Expression Slide (10X Genomics). Standard protocols for the Visium Methanol Fixation and H&E Staining were performed, and each capture area was then imaged using an Olympus VS120 slide scanner at 20X magnification. The tissue sections were then permeabilized for 12-minutes, and poly-adenylated mRNAs were captured by primers on the slide. The protocols of the Visium Spatial Gene Expression Kit were then followed to produce full-length cDNA, and the Visium Library Construction Kit (10X Genomics) was used to generate sequencing libraries according to the manufacturer's instructions. A NovaSeq 6000 Sequencer (Illumina) was used to sequence amplified libraries at approximately 50,000 reads per spot for each capture area covered by tissue based on the instructions of the Visium Spatial Gene Expression manual. RNA libraries were prepared for sequencing using standard Illumina protocols.