Project description:Technical control for allelic detection using Smart-seq3. Liver RNA from pure C57BL6/J and CAST/EiJ strains was combined at varying ratios (0:1, 1:7, 1:3, 3:5, 1:1, 5:3, 3:1, 7:1, 1:0) for a total of 200 pg RNA per sample.
Project description:Missing values in proteomic data sets have real consequences on downstream data analysis and reproducibility. Although several imputation methods exist to handle missing values, no single imputation method is best suited for a diverse range of data sets, and no clear strategy exists for evaluating imputation methods for large-scale DIA-MS data sets, especially at different levels of protein quantification. To navigate through the different imputation strategies available in the literature, we have established a workflow to assess imputation methods on large-scale label-free DIA-MS data sets. We used three DIA-MS data sets with real missing values to evaluate eight different imputation methods with multiple parameters at different levels of protein quantification; dilution series data set, a small pilot data set, and a larger proteomic data set.
Project description:Different sample preparation methods were tested for HeLa proteome analysis. A sample obtained using sodium deoxycholate-based lysis allowed identification of the highest number of proteins. For this sample, a dilution series was acquired in triplicates ranging from 0.2ng to 200ng. All measurements were performed on Bruker timsTOF Pro 2 operated in dia-PASEF mode and analysed library-free using DIA-NN 1.8.
Project description:This data is part of a large-scale platform comparison experiment. Whole mouse and adult mouse colon RNA was mixed at different concentrations (100:0, 75:25, 50:50, 25:75, and 0:100) and distributed across multiple centers for analysis. Eight microarray platforms and RT-PCR were tested for different labeling protocols, amplification protocols, and data preprocessing approaches in order to maximize data intercomparability. The role of universal reference RNA was also examined. Probes of different platforms were matched using gene symbols and/or RefSeq/GenBank accession numbers. Several different normalization procedures were applied. Evaluation criteria included linearity, sensitivity/specificity, and reproducibility within and between platforms, labeling methods, and laboratories. Keywords: dilution series
Project description:We report dynamics of X-chromosome upregulation (XCU) along X-chromosome inactivation (XCI) in mESCs as they differentiate into EpiSCs. F1 hybrid C57BL6/J × CAST/EiJ male and female mESCs were adapted to 2i/LIF and female cells grown in serum/LIF conditions were differentiated using Fgf2 and Activin A for 1, 2, 4 and 7 days to induce random XCI. scRNA-seq was performed using the Smart-seq3 protocol, providing full-length coverage together with molecular counting using UMIs. Allelic resolution is achieved using strain-specific SNPs in the data. We reveal dynamic balancing of X alleles as cells undergo XCI to compensate dosage imbalances between sexes as well as between X and autosomes. Furthermore, we reveal that female naïve mESCs with two active X chromosomes lack XCU on both alleles which has major implications for reprogramming studies. Finally, we estimate allelic transcriptional burst kinetics from the data and find that progressively increased burst frequencies underlies the XCU process.
Project description:We report dynamics of X-chromosome upregulation (XCU) along X-chromosome inactivation (XCI) in mESCs as they differentiate into EpiSCs. F1 hybrid C57BL6/J × CAST/EiJ male and female mESCs were grown in serum/LIF conditions were differentiated using Fgf2 and Activin A for 1, 2, 4 and 7 days to induce random XCI in female cells. Multi-modal single-cell sequencing was performed using scATAC on nuclei and Smart-seq3 to assay chromatin accessibility and poly-A+ RNA expression, respectively. Allelic resolution is achieved using strain-specific SNPs in the data. We reveal dynamic balancing of X alleles as cells undergo XCI to compensate dosage imbalances between sexes as well as between X and autosomes. Furthermore, we reveal that female naïve mESCs with two active X chromosomes lack XCU on both alleles which has major implications for reprogramming studies. Finally, we estimate allelic transcriptional burst kinetics from the data and find that progressively increased burst frequencies underlies the XCU process.
Project description:Allele-sensitive RNA sequencing of single-cells can be used to infer the kinetics of transcriptional bursts in eukaryotic cells. Here, we used the Smart-seq3 protocol to prepare libraries from two 384-well plates of primary mouse fibroblasts. The fibroblasts were derived from tail explants of a male adult mouse (F1 offspring of C57 x CAST cross). The samples were sequenced to high depth using MGI's DNBSEQ G400RS platform using paired-end 100 bp reads.
Project description:We have identified candidate genes from the Feml2 QTL influencing femur length through allele specific expression analysis of growth plates in C57BL/6J x CAST/EiJ F1 hybrids. This work provides the foundation to identify novel genes affecting bone geometry.
Project description:The objective of this project is to study the proteome in different tissues of huntingtin knock-in allelic series in mice. In the current part of the study, heart samples from the complete allelic series collected at 2, 6, and 10 months were analyzed. To this end, comprehensive quantitative, label-free proteome analysis was performed using Evotec’s quantitative Deep Proteome Profiling technology.