GSM9232562: Pancreas, Cpa1, VisiumHD; Mus musculus; OTHER
Pancreata of 8-week-old male Cre and Cpa1 mice were isolated. Mice were euthanized by cervical dislocation, pancreata immediately isolated, rinsed with cold PBS buffer, and processed as described in the histology section. Paraffin blocks were stored at 4°C until further processing. Tissue blocks were incubated on ice bath 20 minutes before sectioning. For each specimen, two sections at 10 µm thickness were cut, and RNA was isolated using Deparaffinization Solution (Qiagen GmbH, 19093), and RNeasy®FFPE Kit for RNA Extraction (Qiagen GmbH, 73504) according to the manufacturer's protocol. RNA quality was assessed by measuring the percentage of total RNA fragments greater than 200 nucleotides (DV200) with the 2100 Bioanalyzer Instrument using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Inc.; 5067-1511). Cre DV200 was 83%, Cpa1 DV200 48%; 10x Genomics recommended minimum requirement 30%. Next, tissue sections at 5 µm thickness were prepared and placed on poly-L-lysine-coated glass slides (Sigma-Aldrich Co., P0425-72EA) and dried for 30 minutes on a heating plate at 42°C, followed by subjecting to a 42°C dry-incubation in an oven for three hours. Subsequently, the sections were stored at room temperature in a desiccator for 24 hours. Library preparation process was conducted in accordance with the manufacturer's protocols (10x Genomics Inc.; Visium HD FFPE Tissue Preparation Handbook, CG000684 and Spatial Gene Expression Reagent Kits User Guide, CG000685), and reagents (Visium HD Spatial Gene Expression Reagent Kit – small, 1000668; and Visium HD Mouse Transcriptome Probes v2 – small, 1000667). In brief, the sections were deparaffinized and H&E stained to assess tissue morphology. For deparaffinization tissue slides were incubated for two-hours at 60°C in an oven, cooled for five minutes at room temperature and deparaffinized by immersing slides twice for 10 minutes in xylene (Sigma-Aldrich, 214736). Next, slides were rehydrated in an ethanol (Fisher Scientific GmbH, 17740239) series consisting of 2 × 100%, 2 × 96%, and 1 × 70% ethanol, with each step lasting three minutes, following incubation in distilled water for 20 seconds. Deparaffinized and rehydrated slides were stained with 1 mL of Gill II hematoxylin solution (Sigma-Aldrich, GHS232) and incubated at room temperature for 30 seconds. Excessive staining solution was removed by immersing the slides in three water beakers. 1 mL of the bluing buffer (Agilent Technologies, Inc., CS70230-2) was added, and slides were incubated for one minute prior to being immersed in a water beaker. Subsequent staining was performed using alcoholic eosin (Sigma-Aldrich, HT110116) by applying 1 ml of eosin for one minute and washing out by immersing the slides in two water beakers. Finally, slides were coverslipped using 85% glycerol (Thermo Scientific Chemicals, 327255000) mounting medium. For imaging Axio Scan Z.1 slide scanner (Carl Zeiss AG) was used, and the area of interest (AOI) was determined. Following the removal of the coverslips, tissue slides were placed in the tissue cassettes (10x Genomics Inc., Visium Tissue Slide Cassette S3, 1000684), and destained to remove residual hematoxylin and eosin. Subsequent to the initial destaining step, samples underwent decrosslinking to enhance the accessibility of probes to target RNA molecules. Then, hybridization was performed using mouse transcriptome specific probes. In the subsequent phase – probe ligation – a ligase was incorporated, resulting in the formation of a ligation product. The cassettes were then maintained at a temperature of 4°C. Visium HD slide (10x Genomics Inc., Visium HD Slide, 1000670) was thawed and prepared prior to the execution of the Visium CytAssist device. Further steps, including probe release and capture, are facilitated within the Visium CytAssist device. Subsequent to being released from the tissue, the ligation products were captured on the Visium slide and extended by the addition of the spatial barcode. The spatially-barcoded ligation products were amplified, followed by SPRIselect cleanup (SPRIselect DNA Size Selection Reagent, Beckman Coulter Inc., B23317). Next, quantitative polymerase chain reaction (qPCR) was performed to establish the sample index PCR cycle number for gene expression libraries. Following amplification, the material was subjected to indexing via sample index PCR, thereby generating library molecules. Subsequent to the cDNA amplification, the libraries were purified using SPRIselect and qualitatively assessed with the 2100 Bioanalyzer Instrument using the High Sensitivity DNA Kit (Agilent Technologies, Inc., 5067-4626). The final libraries were sequenced by Novogene (Munich, Germany) on an Illumina NovaSeq X Plus Series platform in paired-end mode (PE150). Raw data was obtained in FASTQ format.