Project description:To understand gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single-cell level.
Project description:LNPs have been demonstrated to hold great promise for the clinical advancement of RNA therapeutics. Continued exploration of LNPs for application in new disease areas requires identification and optimisation of leads in a high throughput way. Currently available high throughput in vivo screening platforms are well suited to screen for cellular uptake but less so for functional cargo delivery. We report on a platform which measures functional delivery of LNPs using unique peptide ‘barcodes’. We describe the design and selection of the peptide barcodes and the evaluation of these for the screening of LNPs. We show that proteomic analysis of peptide barcodes correlates with quantification and efficacy of barcoded reporter proteins both in vitro and in vivo and, that the ranking of selected LNPs using peptide barcodes in a pool correlates with ranking using alternative methods in groups of animals treated with individual LNPs. We show that this system is sensitive, selective, and capable of reducing the size of an in vivo study by screening up to 10 unique formulations in a single pool, thus accelerating the discovery of new technologies for mRNA delivery.
Project description:We have generated 979 yeast strains in which the natural 3' UTR of essential gene mRNAs has been replaced by the same long 1.4 kb artificial 3' UTR (DAmP modification). Nonsense mediated mRNA decay (NMD) of these mRNA reporters was tested by using Agilent barcode microarrays by taking advantage of molecular barcodes introduced just downstream the stop codon during strain construction. We introduced in each DAmP strain either a neutral mutation (deletion of YEL068C) or the deletion of essential factors for NMD: NAM7 and NMD2. The resulting haploid cells were tested for changes in DAmP mRNA levels by comparing an RNA sample with a DNA genomic sample. Differences between the samples indicate the sensibility of the DAmP mRNAs to degradation through NMD. The large scale results were validated by Q-PCR analysis of individual strains. A strong negative correlation between the level of destabilization elicited by a long 3' UTR and the size of the coding sequence suggests that long ORF mRNAs can escape NMD even in the presence of a long 3' UTR. Three samples were analysed and for each sample a technical replicate was performed, starting from the raw cellular material. YEL068C samples represent the reference population while NMD2 and NAM7 samples are the ones important for the analysis. Only a very limited part of the Agilent barcode microarray signal (barcodes corresponding to the “up” tag in the DamP strain) was used for the interpretation of the data.
Project description:Using microfluidics, well-defined barcodes were generated on the slide surface by cross-amplification, followed by high-throughput sequencing using Novaseq to detect spatial transcriptomic information in the mouse brain.
Project description:Genome wide DNA methylation profiling of blood samples from eight female identical twins of Han Chinese for forensic age prediction, age 21 to 32. The Illumina Infinium HumanMethylation450 BeadChip was used to obtain DNA methylation profiles across approximately 485,000 CpGs at a single-nucleotide resolution. Samples included 8 pairs of identical female twins of Han Chinese.
Project description:Several template DNA molecules with random base molecular barcodes were amplified and sequenced, and the efficacy of the random base barcode for digital counting was shown.