GSM6833486: Patient 3, ST; Homo sapiens; OTHER
All OCT-embedded tissues were sectioned into 10 μm slices and placed on the Capture Area (6.5 mm × 6.5 mm) of Visium Spatial slides (Visium Spatial Gene Expression Slide, 10× Genomics, Pleasanton, CA) by a cryostat microtome (Leica Microsystem). The prepared slides were stored at a dry environment for fixation and staining.  Sections incubated by pre-chilled methanol (Millipore Sigma, Darmstadt, Germany) and isopropanol (Millipore Sigma) were stained by Mayer's hematoxylin (Dako, Agilent, Santa Clara, CA) for 7 min, Bluing buffer (Dako) for 2 min, Eosin Y solution (Millipore Sigma) diluted 1:10 in Tris-base (ThermoFisher Scientific, 0.45 M, pH 6.0) for 1min, respectively. Slides were fully rinsed by Milli-Q water (Corning, Corning, NY) after each staining. Images of all Capture Areas in the fiducial frames were taken in the bright-field and imported into 3DHISTECH software.  After placed in the Slide Cassette, each well of the sections were uniformly covered with 70 μl permeabilization enzyme (10× Genomics). Sections were incubated at 37℃ following a time gradient (3, 6, 12, 18, 24, 30 min). Each well of tissue sections, after the removal of permeabilization enzyme by pipette, were rinsed with 100 μl 0.1× SSC (Sigma-Aldrich, St. Louis, MO). Reverse transcription was conducted by Bio-Rad PCR instrument (MyCycler, Bio-Rad, Hercules, CA) in accordance with the protocol of 10× Genomics (Protocol # CG000238). cDNAs in the supernatant, released during reverse transcription, were collected for second strand synthesis, denaturation, in vitro transcription and adaptor ligation. The products of a second reverse transcription were applied in the construction of spatial transcriptomic libraries, which was sequenced on the Illumina NovaSeq 6000 platform with over 4, 000 spots per sample and over 60, 000 reads per spots.