STOmics_BlockB
Tissue samples were sent to MGI, and STEREOseq was performed in their facility under standard manufacturer-recommended procedures. Pre-frozen OCT tissues were transversely sectioned at 10 ?m thickness (Leica CM1950 cyrostat). Tissue sections were adhered to the Stereo-seq chip surface and incubated at 37 C for 5 minutes. Then, tissues were fixed in methanol and incubated at -20 C for 30 minutes and then dried. Nuclear staining and imaging were performed with ssDNA Qubit dye and fluorescent microscopy (FITC channel). Immediately adjacent tissue sections were collected for histological examination with hematoxylin and eosin. The tissue on the chip was permeabilized, incubated at 37 C, and then washed. Released RNA from permeabilized tissues was captured by the DNB and reverse transcribed overnight at 42 C. After in situ reverse transcription, tissue patches were washed and digested with tissue removal buffer at 55 C for 10 minutes. cDNA-containing chips were then subjected to cDNA release treatment and collection. The resulting cDNAs were amplified and purified using SPRI-bead purification for DNB generation.
SUB15760521
FP200008330