Mus musculus
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ABSTRACT:
PROVIDER: PRJNA387734 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:we describe a streamlined RNAseq protocol (EASY RNAseq) for sensitive transcriptome assessment starting from low amount of input materials. EASY RNAseq is technically robust enough for sequencing small pools of homogenous and heterogeneous cells, recovering higher numbers of genes and with a more even expression distribution pattern than other commonly used methods.
2020-08-01 | GSE135290 | GEO
Project description:Deeper understanding of T cell biology is crucial for the development of new therapeutics. Human naïve T cells have low RNA content and their numbers can be limiting; therefore we set out to determine the parameters for robust ultra-low input RNA sequencing. We performed transcriptome profiling at different cell inputs and compared three protocols: Switching Mechanism at 5’ End of RNA Template technology (SMART) with two different library preparation methods (Nextera (SMART_Nxt) and Clontech (SMART_CC)), and AmpliSeq technology. As the cell input decreased the number of detected coding genes decreased with SMART, while stayed constant with AmpliSeq. However, SMART enables detection of non-coding genes, which is not feasible for AmpliSeq. The detection is dependent on gene abundance, but not transcript length. The consistency between technical replicates and cell inputs was comparable across methods above 1K but highly variable at 100 cell input. Sensitivity of detection for differentially expressed genes decreased dramatically with decreased cell inputs in all protocols, support that additional approaches, such as pathway enrichment, are important for data interpretation at ultra-low input. Finally, T cell activation signature was detected at 1K cell input and above in all protocols, with AmpliSeq showing better detection at 100 cells.
2019-05-24 | GSE130882 | GEO
Project description:Plant small RNAs are a diverse and complex set of molecules, ranging in length from 21 to 24 nt, involved in a wide range of essential biological processes. Nowadays, high-throughput sequencing is the most commonly used method for the discovery and quantification of small RNAs. However, it is known that several biases can occur during the preparation of small RNA libraries, especially using low input RNA. We used two types of plant biological samples to evaluate the performance of seven commercially available methods for small RNA library construction, using different RNA input amounts. We show that when working with plant material, library construction methods have differing capabilities to capture small RNAs, and that different library construction methods provide better results when applied to the detection of microRNAs, phased small RNAs, or tRNA-derived fragments.
2021-08-01 | GSE156973 | GEO
Project description:Here, we report an enrichment-based ultra-low input cfDNA methylation profiling method using methyl-CpG binding proteins capture, termed cfMBD-seq. We optimized the conditions of cfMBD capture by adjusting the amount of MethylCap protein along with using methylated filler DNA. Our data showed that cfMBD-seq performs equally to the standard MBD-seq (>1000 ng input) even when using 1 ng DNA as the input. cfMBD-seq demonstrated equivalent sequencing data quality as well as similar methylation profile when compared to cfMeDIP-seq. We showed that cfMBD-seq outperforms cfMeDIP-seq in the enrichment of CpG islands. This new bisulfite-free ultra-low input methylation profiling technology has a great potential in non-invasive and cost-effective cancer detection and classification.
2021-01-01 | GSE161331 | GEO
Project description:Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq
| PRJNA376510 | ENA
Project description:Background: Identification of locus-locus contacts at the chromatin level provides a valuable foundation for understanding of nuclear architecture and function and a valuable tool for inferring long-range linkage relationships. As one approach to this, chromatin conformation capture-based techniques allow creation of genome spatial organization maps. While such approaches have been available for some time, methodological advances will be of considerable use in minimizing both time and input material required for successful application. Results: Here we report a modified tethered conformation capture protocol that utilizes a series of rapid and efficient molecular manipulations. We applied the method to Caenorhabditis elegans, obtaining chromatin interaction maps that provide a sequence-anchored delineation of salient aspects of Caenorhabditis elegans chromosome structure, demonstrating a high level of consistency in overall chromosome organization between biological samples collected under different conditions. In addition to the application of the method to defining nuclear architecture, we found the resulting chromatin interaction maps to be of sufficient resolution and sensitivity to enable detection of large-scale structural variants such as inversions or translocations. Conclusion: Our streamlined protocol provides an accelerated, robust, and broadly applicable means of generating chromatin spatial organization maps and detecting genome rearrangements without a need for cellular or chromatin fractionation. Application of modified version of TCC protocl using different C. elegans strains (N2 and glp-1) in L1, and adult life stages.
2016-01-20 | E-GEOD-76930 | biostudies-arrayexpress
Project description:Different Library Sample Preparation (LSP) allow the detection of a large common set of isoforms. However, each LSP also detects a smaller set of isoforms which are characterized both by lower coverage and lower FPKM than that observed for the common ones among LSPs. This characteristic is particularly critical in case of low input RNA NuGEN v2 LSP. The effect of statistical detection of alternative splicing considering low input LSP (NuGEN v2) with respect to high input LSP (TruSeq) was studied using a benchmark dataset, in which both synthetic reads and reads generated from high and low input LSPs were spiked-in. Statistical detection of alternative splicing was done using prototypes of bioinformatics analysis for isoform-reconstruction and exon-level analysis. Each available sample contains a total of 5 paired end replicates. 3 samples contain increasing numbers of spiked-in reads (20, 40, 80 millions) from NuGENv2 library preparation kit on a common TruSeq 1000ng background. 3 additional samples were built with the same approach, but spiked-in reads were collected from a TruSeq-based experiment. The remaining 6 samples follow the same approach of the previous 6, but the common background is based on a TruSeq library preparation on 100ng of material
2014-05-28 | E-GEOD-58001 | biostudies-arrayexpress
Project description:Dysregulated protein synthesis is a major underlying cause of many neurodevelopmental diseases such as Fragile X Syndrome. A very robust technique is required to capture subtle but biologically significant differences in neurological disorders. Ribosome profiling, which is based on deep sequencing of mRNA fragments protected from ribonuclease digestion by ribosomes, is a powerful tool to study translational control. However, it has been mainly applied to rapidly dividing cells where translation is robust and where large amounts of starting material are readily available. The application of ribosome profiling to low-input brain tissue where translation is modest and where gene expression changes between genotypes are expected to be small has not been carefully evaluated. Using hippocampal tissue from wide type and fragile X mental retardation 1 (Fmr1) knockout mice, we show that variable RNase digestion can lead to significant sample batch effects. We also establish GC content and ribosome footprint length as quality control metrics for ribonuclease digestion. We performed ribonuclease titration experiments for low-input samples to identify optimal conditions for this critical step that is often improperly conducted. Our data reveal that optimal RNase digestion is essential to ensure high quality and reproducibility of ribosome profiling especially for low-input brain tissue.
2019-03-15 | GSE116233 | GEO
Project description:Different Library Sample Preparation (LSP) allow the detection of a large common set of isoforms. However, each LSP also detects a smaller set of isoforms which are characterized both by lower coverage and lower FPKM than that observed for the common ones among LSPs. This characteristic is particularly critical in case of low input RNA NuGEN v2 LSP. The effect of statistical detection of alternative splicing considering low input LSP (NuGEN v2) with respect to high input LSP (TruSeq) was studied using a benchmark dataset, in which both synthetic reads and reads generated from high and low input LSPs were spiked-in. Statistical detection of alternative splicing was done using prototypes of bioinformatics analysis for isoform-reconstruction and exon-level analysis.
2014-05-28 | GSE58001 | GEO
Project description:Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This hampers the investigation of chromatin structure in rare cell populations. We present two new low-input Capture-C protocols that generate high-quality, reproducible interaction profiles from fewer than 20,000 cells, and show that these are not biased by PCR amplification or the degree of formaldehyde fixation.
2017-11-20 | GSE99257 | GEO