title: GSM9065345: Primary tumour, MDA-MB-231 xenograft model, Mouse 2; Homo sapiens; Mus musculus; OTHER
library_name: GSM9065345
library_construction_protocol: Cryo-sectioning was done on OCT embedded and snap frozen tissue samples at 10um thickness and placed on cold Visium slide arrays. The sections were adhered by swiftly warming of the backside of the slide. The slides were then kept in -80°C less than 4 weeks before processing accordingly. In short, the slides were first warmed at 37°C for one minute and then immersed in pre-chilled methanol (VWR EU, 20847.307) for 30 minutes at -20°C for fixation. Staining with hematoxylin and eosin was carried out by one min of drying of the tissues with 500uL of isopropanol (Fisher Scientific, A461-1) followed by air drying until sections turned white. Around 1 mL of with Mayer's hematoxylin (Agilent, S23309) was pipetted onto the slides and treated for four minutes. The slides were then washed in nuclease free water followed by a two-minute incubation with bluing buffer (Agilent, CS702), washed again and then counterstained with buffered eosin (Sigma-Aldrich, HT110216, 1:10 dilution in Tris-Acetic Acid Buffer). The slides were air dried for about 2 minutes and then warmed for 5 minutes at 37°C before mounting using 85percent Glycerol (Merck, 104094) and a coverslip. Bright field histology images were obtained using a 20X objective on Zeiss microscope using the Metafer VSlide system and the images were processed by the VSlide software. The images were extracted as jpgs for downstream analysis. After imaging, the coverslip and the remaining glycerol was washed off in Milli-Q water and the slides were attached in plastic cassettes included in the reagent kit and first subjected to 20 minutes of permeabilization at 37°C to let the mRNA reach the probes on the slide surface for binding. The protocol was then followed without deviations to create amplified libraries which in the end were individually indexed using the Dual Index Kit TT SetA, (PN-1000215, 10X Genomics), quality controlled on a BioAnalyzer instrument and concentrations were measured using Qubit DNA HS. The libraries were pooled equimolarly (2nM) and sequenced on the Nextseq 2000 (Illumina platform). To reach the appropriate read depth the recommended number of reads per ST spot were applied according to the protocol. Spatial transcriptomics was conducted using 10X Visium Spatial Gene Expression Slide and Reagent Kit, 16 rxns (PN-1000184), according to the protocol detailed in document CG000239RevD for the TNBCs and CG000239RevE for the Xenografts, available in 10X Genomics demonstrated protocols.
identifiers.namespace: GEO
identifiers.id: GSM9065345_r1