Project description:This dataset consists of two individual sample-multiplexing (MULTI-seq) single-cell RNA sequencing experiments, MB10x01 and MB10x02. Single-cell RNA sequencing (10X Genomics) analyses were performed on a microfluidic 3D in vitro blood-brain-barrier model (containing primary human brain microvascular endothelial cells, brain vascular pericytes, and astrocytes) perfused with P. falciparum egress product (MB10x01) or P. falciparum-infected red blood cells (RBC) (MB10x02). Dataset MB10x01 included two samples multiplexed by MULTI-seq sample barcoding (TCCTCGAA for control RBC lysate, ATGCGATG for P. falciparum egress product). P. falciparum egress product was obtained by letting tightly synchronized P. falciparum-infected RBC egress in media used for perfusions (5x10^7 infected RBC/ml). 3D blood-brain-barrier models perfused with P. falciparum egress products were incubated for 24 hours and compared to a control perfused with uninfected red blood cell lysate. MULTI-seq barcoding (McGinnis et al.Ê2019) was used for sample-barcoding of these two conditions, and the dataset contains cDNA (transcriptome) and sample barcode read files. Dataset MB10x02 included three samples multiplexed by MULTI-seq sample barcoding (GCTATGCA for control RBC, CGATACTG for Trophozoite stage, TACGCAGT for Schizont stage). 3D blood-brain-barrier models were perfused for 30 minutes with P. falciparum-infected RBC in the Trophozoite stage (26-34 hours post invasion) or Schizont stage (42-48 hours post invasion) (5x10^7 infected RBC/ml). After a 20-minute wash, the 3D blood-brain-barrier models were incubated with the bound P. falciparum-infected RBC for 6 hours and compared to uninfected RBC perfused controls. MULTI-seq barcoding was used for sample-barcoding of the three conditions, and the dataset contains cDNA (transcriptome) and sample barcode read files.
Project description:Cellular barcoding using heritable synthetic barcodes coupled to high throughput sequencing is a powerful technique for the accurate tracing of clonal lineages in a wide variety of biological contexts. Recent studies have integrated cellular barcoding with a single-cell transcriptomics readout, extending the capabilities of these lineage tracing methods to the single-cell level. However there remains a lack of scalable and standardised open-source tools to pre-process and visualise both bulk and single-cell level cellular barcoding datasets. Here, we describe bartools, an open-source R-based toolkit that streamlines the pre-processing, analysis and visualisation of synthetic cellular barcoding datasets. In addition, we developed BARtab, a portable and scalable Nextflow pipeline that automates upstream barcode extraction, quality control, filtering and enumeration from high throughput sequencing data. In addition to population-level cellular barcoding datasets, BARtab and bartools contain methods for the extraction, annotation, and visualisation of transcribed barcodes from single-cell RNA-seq and spatial transcriptomics experiments, thus extending the analytical toolbox to also support novel expressed cellular barcoding methodologies. We showcase the integrated BARtab and bartools workflow through the analysis of bulk, single-cell, and spatial transcriptomics cellular barcoding datasets.
Project description:Detecting strain-specific barcodes with mass spectrometry can facilitate the screening of genetically engineered bacterial libraries. Here, we introduce intact protein barcoding, a method to measure protein-based library barcodes and metabolites using flow-injection mass spectrometry (FI-MS). Protein barcodes are based on ubiquitin with N-terminal tags of six amino acids. We demonstrate that FI-MS detects intact ubiquitin proteins and identifies the mass of N-terminal barcodes. In the same analysis, we measured relative concentrations of primary metabolites. We constructed 6 ubiquitin-barcoded CRISPRi strains targeting metabolic enzymes, and analyzed their metabolic profiles and ubiquitin barcodes. FI-MS detected barcodes and distinct metabolome changes in CRISPRi-targeted pathways. We demonstrate the scalability of intact protein barcoding by measuring 132 ubiquitin barcodes in microtiter plates. These results show that intact protein barcoding enables fast and simultaneous detection of library barcodes and intracellular metabolites, opening up new possibilities for mass spectrometry-based barcoding.