GSM6697345: cartilage_mutant (DSP-1012500008771-F-A08); Mus musculus; OTHER
FFPE mouse bone samples were baked overnight at 37°C followed by 3 hours of baking at 65°C, then loaded onto a Leica Bond RX Fully Automated Research Stainer for subsequent processing steps. The processing protocol included three major steps: 1) slide baking, 2) antigen retrieval for 20min at 100°C, and 3) treatment with Proteinase K (1.0 µg/mL in 1xPBS) for 15min. Following these steps, slides were removed from the Leica Bond RX, and a cocktail of GeoMx Mouse Whole Transcriptome Atlas probes (targeting over 19,000 targets) were applied to each slide and allowed to hybridize at 37°C overnight in a humidity chamber. The following day, slides were washed, blocked, and allowed to incubate with a combination of Alexa Fluor 594 Anti-alpha smooth muscle Actin antibody (Abcam antibody ab202368; clone: 1A4) and Syto83 nucleic acid stain. Slides were stained for 1h at room temperature in a humidity chamber. Slides were then washed and loaded onto the GeoMx instrument. On the GeoMx machine, slides were fluorescently scanned, and ROIs were manually placed in the following areas: trabecular bone and cartilage. The GeoMx device exposed ROIs to 385 nm light (UV), releasing the indexing oligos. Indexing oligos were collected with a microcapillary and deposited into a 96-well plate. Samples were dried down overnight, then resuspended in 10 µL of DEPC-treated water. PCR was performed using four µL of each sample and the oligos from each ROI were indexed using unique i5 and i7 dual-indexing systems (Illumina). PCR reactions were purified twice using AMPure XP beads (Beckman Coulter, Inc) according to the manufacturer's protocol. Purified libraries were sequenced on an Illumina NovaSeq 6000 according to the manufacturer's instructions, with 27x27 pair end reads.