GSM9038419: Stereo-seq_Opossum; Monodelphis domestica; OTHER
Animals were deeply anesthetized with isoflurane followed by decapitation, and brains were immediately removed and submerged in ice-cold 1x PBS containing RNase inhibitor (1 unit per µL; Thermo Fisher Cat. # AM2696). Extracted brains were placed in an ice-cold metal mold (opossum: Kent Scientific Cat. # RBMS-300C; mouse: RBMS-205C) and in the opossum, 2 mm coronal slices were obtained by bisecting the superior colliculus with one blade and inserting additional blades 2 mm anterior and posterior to the first (Wong and Kaas, 2009). In the mouse, one blade was inserted at the cortical margins of the midline (below the lambdoid suture) and additional blades 1 mm anterior and posterior to the first (Wang et al., 2020). These slices were immediately removed from the mold and placed back into ice-cold PBS. To embed two coronal slices (containing four total V1 hemispheres) in the opossum, cuts were made lateral to V1 (~3 mm from the midline) in both hemispheres, as well as horizontal (~4 mm from the highest point of cortical surface). Because of the difference in brain size, these cuts were not necessary in the mouse. Coronal slices were arranged in an ice-cold metal base mold (Azer Scientific Cat. # ES4440) containing a drop of OCT (Tissue-Tek), then covered in OCT and flash-frozen in liquid nitrogen. Blocks were stored at -80°C and sectioned at -20°C. The Stereo-seq pipeline was performed according to user manuals provided by the vendor (Complete Genomics). Sectioning and imaging was performed by the UCLA Translational Pathology Core Laboratory (TPCL). RNA integrity (RIN) analysis, permeabilization, library preparation, and sequencing was performed by the UCLA TCGB. First, RNA integrity numbers (RINs) were obtained from shavings of the embedded tissue blocks. Due to skull thickness and the difficulty of removing the brain quickly in opossums, we obtained a RIN of 7.1 for opossum tissue and a RIN of >9 for mouse tissue. Despite this disparity, both were well above the RIN threshold of 4.0 required for Stereo-seq v1.3. Next, the optimal tissue permeabilization length for each sample was determined according to the Stereo-seq Permeabilization Set for Chip-on-a-slide User Manual. Briefly, four 10-µm sections were cut and mounted onto separate 10 mm x 10 mm chips on the Stereo-seq Chip P slide using the 'warm' method. Chip P slides were then dried, fixed in pre-cooled methanol for 30 minutes, and re-dried. 1X Permeabilization Reagent Solution was pre-warmed to 37°C and applied to different chips for 6, 12, 18, or 24 minutes. Following a wash, Chip P slides underwent in situ reverse transcription. RT QC Mix was added to each chip and incubated at 45°C for 60 minutes. Then, Tissue Removal Mix was incubated for 60 minutes and the slide was washed until no tissue traces remained on the chips. Slides were imaged at 10x magnification using an EVOS™ M7000. The optimal permeabilization time was independently chosen to be 12 minutes for both mouse and opossum samples based on maximal fluorescence signal with minimal signal diffusion. Next, the transcriptomics procedure was performed according to the Stereo-seq Transcriptomics Set for Chip-on-a-slide User Manual. First, a single 10-µm section was cut and mounted onto the 10 mm x 10 mm chip on the Stereo-seq Chip T slide. Chip T slides were then dried, fixed in pre-cooled methanol for 30 minutes, and re-dried before staining with nucleic acid dye (Qubit ssDNA Assay Kit, Invitrogen Cat. # Q10212) and imaging at 10x magnification (EVOS M7000; fluorescein isothyocyanate channel). Chip T slides then underwent tissue permeabilization (12 minutes) and in situ reverse transcription before cDNAs were released, denatured, amplified, and purified. The resulting libraries were sequenced on a full MGI DNBSEQ-T7 flow cell with paired-end, 75 bp reads.