GSM9555589: Patient 062, WD component; Homo sapiens; RNA-Seq
Fresh tissue was harvested at the time of surgery from both WD and DD components and stored immediately in liquid nitrogen. Additional tissue from both components was placed in formalin for permanent fixation and then embedded in paraffin. Tumor diagnosis and subtype was validated by expert histological review and DNA FISH (Empire Genomics, SKU MDM2-CHR12-20-GROR) was used to confirm the presence of MDM2 amplification (authors SD, MN). Tumors without MDM2 amplification were excluded. After tumor diagnosis and subtype were confirmed, samples were thawed in RNALaterTM-ICE (ThermoFisher) overnight at -20C. Nuclei were then isolated using the Chromium Nuclei Isolation with RNase Inhibitor Kit (10X Genomics). For DD components, 50mg of tissue was used and for WD components, 250mg of tissue was used from each sample. Nuclei concentration and cell viability was determined using a Countess II FL Automated Cell Counter (ThermoFisher). Cell viability <5% was used as a threshold to ensure high quality nuclei isolation. Samples were also assessed by brightfield microscopy for the presence of significant debris. Single nuclei suspensions generated from each sample were then used to construct 3'GEX cDNA libraries (10x Genomics). Single nuclei suspensions generated from each sample were used to construct 3'GEX cDNA libraries (10x Genomics) followed by next-generation sequencing via NovaSeq 6000 (Illumina). Demultiplexed sequencing results were aligned to the reference genome and ambient RNA was removed using CellBender. After filtering, 27,333 nuclei were included in the computational analysis pipeline. The R package Seurat was applied to integrate samples, cluster cells, and identify differentially expressed genes. LPS clusters were analyzed by pseudo-time analysis (Monocle3) to establish trajectory inferences between WD and DD subtypes.