Project description:We profile single cells from patients with colorectum, ovary and breast cancer using various single-cell technologies, including Chromium 3’ and 5’ single-cell RNA-sequencing. <br>Note: Raw data files have been removed upon submitter's request.
Project description:Single-cell RNA sequencing was performed on retinal tissue from 12-week-old wild-type and Akimba (Ins2AkitaxVEGF+/-) mice, which are known to replicate features of clinical diabetic retinopathy. The aim of this study was to provide deeper insight into the complex network of molecular and cellular changes that underlie diabetic retinopathy by measuring the transcriptional changes that occur in the different cellular compartments of the degenerating diabetic mouse retina. Retinas (n=4 for Akimba, n=2 for wild-type) were isolated in ice-cold Dulbecco’s Modified Eagle Medium. After rinsing with Dulbecco’s Phosphate-Buffered Saline containing 2% fetal bovine serum, each retina was incubated with 1mL digestion buffer (2mg/mL collagenase-P, 200U/mL DNAse-I (Sigma-Aldrich) in M199 medium (Life Technologies) at 37°C for 10min. Retinal tissue was further dissociated by trituration and the suspension was filtered through a 40µm cell strainer and centrifuged for 5min at 300xg (4°C). Pooled retinal single-cell suspensions from wild-type and Akimba were counted on a Luna-FL Cell Counter (Logos Biosystems) and libraries were prepared with the Chromium Single-cell 3’ V2 Chemistry Library Kit, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics) aiming for 5000 cells per library. Barcoded libraries were sequenced on an Illumina HiSeq4000 in 25-8-98 paired-end configuration. The transcriptome data for 9474 retinal cells were analysed, yielding 15 clusters corresponding to eight distinct retinal cell types.
Project description:Single cell RNA-sequencing has been applied to core and border regions of 9 colorectal tumors as well as to matched adjacent non-malignant colon tissue for the purpose of generating a cellular map of colorectal tumors and their tumor microenvironment.
Project description:Pancreatic neuroendocrine neoplasms (PNENs) are biologically and clinically heterogeneous neoplasms. We used quantitative global proteomic analysis on 40 PNENs to compliment paired transcriptome data.
Project description:Neuroblastoma is a tumour of the sympathetic nervous system, with a clinical phenotype resulting from complex patterns of genetic abnormalities, accounting for the most common extracranial neoplasia in childhood. Here we report 44 copy number variations, validated by qPCR, in paired tumour and blood samples of seven patients with neuroblastoma. Thirty four genes were included in altered regions while 11 altered regions did not apparently contain known genes. Five changes were present both in tumour and blood DNA samples in four patients, suggesting presence of germline and/or somatic changes maintained throughout tumorigenesis. Furthermore, some similar or identical changes were observed in different patients. The X chromosome showed the largest number of alterations. DNA from peripheral blood and tumour samples was isolated following using Qiagen DNA Blood Mini Kit. Microarray assays were performed in an Affymetrix platform with Cytogenetics Whole-Genome 2.7 M Array® (Affymetrix, Inc., Santa Clara, CA) for detecting copy number variation across coverage of the whole genome with the highest density of markers for superior resolution. Assays were performed following the procedure of Cytogenetics Copy Number Assay User Guide (Affymetrix®) and analysed with the Affymetrix® Chromosome Analysis Suite (ChAS) software. Gains and losses in tumour and blood samples from each patient were estimated with 95% confidence intervals.
Project description:SWATH-MS enables accurate and reproducible label-free quantification of proteomes including in extensively studied model organisms such as the mouse. Here we present a comprehensive mouse reference spectral library (MouseRefSWATH) that allows quantification of up to 10,597 proteins (62.2% of mouse proteome) by SWATH-MS. We exploit MouseRefSWATH to develop an analytical pipeline for species-specific deconvolution of proteomic alterations in human tumour xenografts (XenoSWATH). This method overcomes the challenge of high protein sequence similarity between the mouse and human, facilitating the study of host microenvironment-tumour interactions from ‘bulk tumour’ measurements. We apply XenoSWATH to characterise a xenograft model of breast ductal carcinoma in-situ and uncover complex regulation of cell migration pathways that is not restricted to the tumour cells but also operates in the mouse stromal upon progression to invasive disease. MouseRefSWATH and XenoSWATH opens new possibilities for in-depth proteomic assessment to address wide-ranging biological questions involving this important model organism.
Project description:We have sampled several tumour regions from a single patient before and after everolimus treatment, including both primary and metastatic site samples to investigate intra-tumour heterogeneity. We selected 4 primary tumour regions, including a pre-treatment biopsy, and 1 metastatic region for expression analysis using microarray data. Two samples were used per region (1 fresh frozen and 1 formalin-fixed).