title: GSM8766751: B16F10 cells treated with Cisplatin, CD45+, gH2Ax+ area, replicate 3; Mus musculus; OTHER
library_name: GSM8766751
library_construction_protocol: Tumors were collected 6 days after the start of the treatment and fixed using 10% neutral buffered formalin for 1 month. Replicate tumors were embedded on the same paraffin block and sliced into 5 µm thick slides. Slides were then immunoprobed for gH2Ax, Pros1 or assayed for Mer-Stat1 binding using Sigma Millipore PLA probes to determine the regions of interest (ROI). Slides were deparaffined, rehydrated and incubated with proteinase K to unmask the cellular RNA targets. Tumors were then treated overnight with RNA Murine Whole Transcriptome Atlas (WTA) probes from Nanostring Technologies to detect the exposed RNAs. Slides were then incubated with Alexa fluor 532 labeled PanCK and Alexa fluor 594 labeled CD45 antibodies. Slides were loaded into the GeoMx DSP instrument following the manufacturer's instructions. 4 representative regions of interest (positive for gH2Ax staining, positive for Pros1 staining, positive for Mer-Stat1 binding or negative for the three previous probes) were selected in each tumor section, manually separating CD45+ and PanCK+ areas inside each picked region. Oligos were then cleaved and collected in a DSP collection plate supplied by Nanostring Technologies. Libraries were prepared using Nanostring Seq code plates C and D and pooled for sequencing using Illumina NovaSeq 600
identifiers.namespace: GEO
identifiers.id: GSM8766751_r1