title: GSM7306435: 10_day, scRNAseq (spatial); Mus musculus; RNA-Seq
library_construction_protocol: For ST transcriptional analysis, dissected thyroid cartilage area tissues from two mice were gently washed with cold phosphate-buffered saline (PBS) (Gibco, catalog no. 20012-043). Then the tissues were placed in OCT-filled mold and snap-frozen in isopentane and liquid nitrogen. Tissue blocks were stored at −80 °C for a maximum of 1 month before use. The Visium Spatial Gene Expression Slide (10 × Genomics, PN-1000185, Pleasanton, CA, USA) includes four capture areas (6.5 mm × 6.5 mm), each capture area has ~5000 gene expression spots, each spot with primers that include Illumina TruSeq Read 1 (partial read 1 sequencing primer), 16-nucleotide (nt) spatial barcode (all primers in a specific spot share the same spatial barcode), 12-nt UMI; 30-nt poly(dT) sequence (captures polyadenylated mRNA for cDNA synthesis) After bright-field imaging, the thyroid ST slide was permeabilized at 37 °C for 24 minutes and washed with 100 μl of 0.1× SSC buffer. Then the on-slide reverse transcription (RT) reaction was performed at 53 °C for 2 hours. Second strand synthesis was subsequently performed on-slide for 15 minutes at 65 °C. Following second strand synthesis, thyroid sample was transferred to tubes containing tris-HCl (1 M; pH 7.0) for amplification and library construction. Library quality was assayed using 1 μl sample on a Bioanalyzer High Sensitivity chip (Agilent, catalog no. 50674626). 10× Genomics Visium libraries were prepared and sequenced on Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) using a 28 bp + 120 bp paired-end sequencing mode. Sequencing data was processed using the Space Ranger pipeline v.1.0.0 (10 × Genomics). This generated data sets containing 1128 spots.