Project description:HCC827 cells were barcoded using the ClonTracer lentiviral barcode library such that the majority of cells were infected with a single barcode. One million cells were expanded to ~120 million cells and split into 8 HYPERfasks. Two HYPERfasks were grown under DMSO and grown until confluence. In six HYPERfasks cells were grown under a GI90 concentration of one of two different inhibitors, gefitinib and trametinib (3 HYPERfasks each). Cells achieved confluence at 4 and 9 weeks for gefitinib and trametinib respectively. During this time, the medium and inhibitor were replenished weekly and DNA was extracted from the medium to track barcode content from dying cells.
Project description:This project investigates hydraplexing, a chemical strategy that increases the detectable fragment-ion signal from labeled analytes in mass spectrometry (MS). The approach introduces several isobaric barcodes onto the same molecule, so that fragmentation yields multiple tag-derived fragments whose intensities accumulate. Using a tetra-amine scaffold derivatized with 2, 3, or 4 TMT tags, we characterized fragmentation patterns under higher-energy collisional dissociation (HCD), determined HCD energy settings that balance precursor transmission with efficient barcode release in MS2, and assessed quantitative performance across direct infusion, liquid chromatography (LC), and capillary electrophoresis (CE) workflows. We further examined performance in a complex environment by analyzing hydraplexed standards spiked into a single-cell Xenopus laevis digest. These experiments demonstrate that fragment-level signal summation can improve quantitative sensitivity in metabolomics- and proteomics-style analyses and suggest routes for extending multi-barcode architectures to broader MSⁿ applications.
Project description:A widespread assumption for single-cell analyses specifies that one cell’s nucleic acids are predominantly captured by one oligonucleotide barcode. However, we show that ~13-21% of cell barcodes from the 10x Chromium scATAC-seq assay may have been derived from a droplet with more than one oligonucleotide sequence, which we call “barcode multiplets”. We demonstrate that barcode multiplets can be derived from at least two different sources. First, we confirm that approximately 4% of droplets from the 10x platform may contain multiple beads. Additionally, we find that approximately 5% of beads may contain detectable levels of multiple oligonucleotide barcodes. We show that this artifact can confound single-cell analyses, including the interpretation of clonal diversity and proliferation of intra-tumor lymphocytes. Overall, our work provides a conceptual and computational framework to identify and assess the impacts of barcode multiplets in single-cell data.
Project description:Barcode-based multiplexing methods can be used to increase throughput and reduce batch effects in large single-cell genomics studies. To evaluate methods for demultiplexing barcode-multiplexed data, we generated a dataset by labeling samples separately with barcode-tagged antibodies, mixing those samples, and progressively overloading a droplet-based scRNA-seq system.