title: GSM8800679: Prrx1-Cre E14.5 biol rep 2; Mus musculus; OTHER
library_construction_protocol: Tissues were fixed in 10% NBF, embedded in paraffin, and sectioned at 5 μm. For GeoMx WTA profiling, slides were deparaffinized and rehydrated, underwent antigen retrieval and Proteinase K digestion, then were hybridized overnight with UV-photocleavable, barcoded oligonucleotide probes from the Mouse Whole Transcriptome Atlas (WTA) RNA probe set. After stringent washing, slides were incubated with Sox9 (EMD-Millipore) and SYTOX13 (NanoString) to allow for visualization of tissue morphology for ROI selection. Slides were placed in the GeoMx Digital Spatial Profiler instrument and ROIs were selected. These selected areas then underwent barcode cleavage from the hybridized probes, with the barcodes collected into individual wells of the collection plate based on ROI of origination. PCR was used to uniquely index each ROIs barcodes with specific Illumina i5/i7 dual indexing primers. These library products were then pooled, purified, and assessed for quality. Paired-end sequencing was performed on a NextSeq 2K P1 PE50 (Illumina), targeting the NanoString recommended read depth of 86 million read-pairs (based on ROI summed areas).