Project description:We conducted a comparison of scRNA-seq on four paired fresh and cryopreserved atheroma samples, two from coronary arteries and two from carotid arteries, to determine whether it is possible to obtain comparable data from frozen samples. After enzymatic digestion into single cell suspensions, we sorted CD45+ cells from half of each sample and immediately processed these using the 10X Genomics scRNA-seq workflow after collection, while the other half was frozen in liquid nitrogen. After cryopreservation for an average of 7 days, the other half of each sample was thawed and sorted identically to the fresh samples.
Project description:We compared the global DNA methylation levels of hairless mouse epidermis using the Infinium Mouse Methylation BeadChip from Illumina and whole genome bisulfite sequencing (WGBS). We also studied the effect of sample storage conditions by using fresh and fresh-frozen epidermis. The DNA methylation levels of xxx,xxx CpG sites covered by both the BeadChip and WGBS were compared. DNA methylation levels obtained with WGBS and the BeadChip were strongly correlated (Pearson correlation r = 0.984). We applied a threshold of 15 reads for the WGBS methylation analysis. Even at a threshold of 10 reads, we observed no substantial difference in DNA methylation levels compared with that obtained with the BeadChip. The DNA methylation levels from the fresh and the fresh-frozen samples were strongly correlated when analysed with both the BeadChip (r = 0.996) and WGBS (r = 0.994). We conclude that the two methods of analysis generally work equally well for studies of DNA methylation of mouse epidermis and find that fresh and fresh-frozen epidermis can generally be used equally well. The choice of method will depend on the specific study’s aims and the available resources in the laboratory.
Project description:Furan is a mouse and rat hepatocarcinogen. We sought to determine if furan-induced gene expression changes could be detected in paired fresh-frozen and formalin-fixed paraffin embedded (FFPE) samples using one-colour microarrays. All samples in this study (fresh-frozen, 18 hours in formalin, 3 weeks in formalin) were also examined using two-colour microarrays and RNA-seq (ribo-depletion and polyA-enrichment protocols) in order to determine the effect of the technology on gene expression profiles.
Project description:Furan is a mouse and rat hepatocarcinogen. We sought to determine if furan-induced gene expression changes could be detected in paired fresh-frozen and formalin-fixed paraffin embedded (FFPE) samples using RNA-seq (polyA-enrichment protocol). All samples in this study (fresh-frozen, 18 hours in formalin, 3 weeks in formalin) were also examined using one- and two-colour microarrays and RNA-seq (ribo-depletion protocol) in order to determine the effect of the technology on gene expression profiles.
Project description:Furan is a mouse and rat hepatocarcinogen. We sought to determine if furan-induced gene expression changes could be detected in paired fresh-frozen and formalin-fixed paraffin embedded (FFPE) samples using RNA-seq (ribo-depletion protocol). All samples in this study (fresh-frozen, 18 hours in formalin, 3 weeks in formalin) were also examined using one- and two-colour microarrays and RNA-seq (polyA-enrichment protocol) in order to determine the effect of the technology on gene expression profiles.
Project description:The aim of the present study was to perform an additional global miRNA microarray analysis of tubular and tubulovillous adenoma biopsy specimen completed with colorectal adenocarcinomas using fresh frozen tisse
Project description:We compared the global DNA methylation levels of hairless mouse epidermis using the recently released Infinium Mouse Methylation BeadChip from Illumina and whole genome bisulphite sequencing (WGBS). We also studied the effect of sample storage conditions by using fresh and fresh-frozen epidermis. The DNA methylation levels of 123,851 CpG sites covered by both the BeadChip and WGBS were compared. DNA methylation levels obtained with WGBS and the BeadChip were strongly correlated (Pearson correlation r = 0.984). We applied a threshold of 15 reads for the WGBS methylation analysis. Even at a threshold of 10 reads, we observed no substantial difference in DNA methylation levels compared with that obtained with the BeadChip. The DNA methylation levels from the fresh and the fresh-frozen samples were strongly correlated when analysed with both the BeadChip (r = 0.999) and WGBS (r = 0.994). We conclude that the two methods of analysis generally work equally well for studies of DNA methylation of mouse epidermis and find that fresh and fresh-frozen epidermis can generally be used equally well. The choice of method will depend on the specific study's aims and the available resources in the laboratory
Project description:Single cell genomics is essential to chart tumor ecosystems. While single cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 212,498 cells and nuclei from 39 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate, and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.
