title: GSM6505136: B16, day 10, mouse 1, rep C, experiment 2; Mus musculus; OTHER
library_construction_protocol: Rat colons were collected from 6-8 week-old Sprague-Dawley rats and were rinsed with ice-cold PBS before O.C.T. or FFPE embedding. Mouse syngeneic tumors were collected in ice-cold PBS before O.C.T. embedding. O.C.T. embedding was accomplished by placing tissue in a cryomold with ice-cold TissueTek O.C.T. Compound on an aluminum block pre-cooled in a dry ice ethanol bath. FFPE tissues were first placed in 10% neutral buffered formalin for 24-hour fixation, after which tissue was processed for embedding in a Sakura VIP automated system with vacuum/pressue cycles, dehydrating in graded alcohols to xylene and then paraffin, and embedded into blocks for sectioning.  All tumor resection smples were obtained with informed concesnt from BioIVT and Discovery Life Sciences. The Donor-A tumor (BioIVT) was divided prior to fixation, one half was O.C.T. embedded, the other half was FFPE embedded. All mouse B16F10 and MC38 samples, and fresh-frozen (FF) rat colon and Donor-A PDAC samples were processed with the Visium Spatial Gene Expression kit (10X Genomics, PN-1000184) according to manufacturer protocol (10X Genomics, CG000160 Rev A and CG000238 Rev D for sectioning/staining and library preparation, respecitvely). Rat colon FF samples with a coverslip (wCS) were coverslipped during imaging, according to manufacturer protocol (10X Genomics, CG000160 RevA). Rat colon and Donor-A FFPE polyA-capture (FFPE-pA) samples were processed as described in Villacampa, et al., Cell Genomics 2021, using reagents from the Visium Spatial gene Expression kit (10X Genomics, PN-1000184), with a modified permeabilization, using 2X permeabilization enzyme for 60 minutes (permebailization enzyme was dissovled at 4 mg/ml (4X) in 0.1 N HCl and diluted to 2X in 0.1 N HCl). Donor A-C FFPE probe samples (FFPE-probes) were processed according to the Visium FFPE Regeant kit with Human Transcriptome Probe kit (10X Genomics, PN-100362 and PN-1000364) according to manufacturer protocol (10X Genomics, CG000409 Rev A and CG000407 Rev A for sectioning/staining and library preparation, respectively).