Single cell RNA-seq by mostly-natural sequencing by synthesis
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA810422 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR20002533.fastq.gz | Fastqsanger.gz | |||
| SRR20002534_1.fastq.gz | Fastqsanger.gz | |||
| SRR20002534_2.fastq.gz | Fastqsanger.gz | |||
| SRR20002535_1.fastq.gz | Fastqsanger.gz | |||
| SRR20002535_2.fastq.gz | Fastqsanger.gz |
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Project description:Here we introduce a mostly natural sequencing-by-synthesis (mnSBS) method for single-cell RNA sequencing (scRNA-seq), adapted to the Ultima genomics platform, and systematically benchmark it against current scRNA-seq technology. mnSBS uses mostly natural, unmodified nucleotides and only a low fraction of fluorescently labeled nucleotides, which allows for high polymerase processivity and lower costs. We demonstrate successful application in four scRNA-seq case studies of different technical and biological types, including 5' and 3' scRNA-seq, human peripheral blood mononuclear cells from a single individual and in multiplex, as well as Perturb-Seq. Benchmarking shows that results from mnSBS-based scRNA-seq are very similar to those using Illumina sequencing, with minor differences in results related to the position of reads relative to annotated gene boundaries, owing to single-end reads of Ultima being closer to gene ends than reads from Illumina. The method is thus compatible with state-of-the-art scRNA-seq libraries independent of the sequencing technology. We expect mnSBS to be of particular utility for cost-effective large-scale scRNA-seq projects.
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Project description:To profile the cell states of interacting natural killer (NK) cells and blood cancer cells, we cultured 26 different cell lines representing diverse hematologic neoplasms either alone or with NK cells derived from three healthy human donors. After 24 h co-culture, we labeled the cells from each monoculture or co-culture condition with oligonucleotide-conjugated antibodies against ubiquitously expressed surface proteins (with different oligonucleotide for each mono- or co-culture), enabling multiplexing in the scRNA-seq using the cell hashing method. We additionally performed pooled CRISPR screens with a single-cell transcriptome readout using the CROP-seq platform in blood cancer cells cultured either alone or in the presence of NK cells to study the effects of perturbing genes that influenced sensitivity to NK cell killing in genome-scale CRISPR screens.
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Project description:We generated single-cell transcriptomes from a large number of single cells using several commercially available platforms, in both microliter and nanoliter volumes, and compared performance between them. We benchmarked each method to conventional RNA-seq of the same sample using bulk total RNA, as well as to multiplexed qPCR, which is the current gold standard for quantitative single-cell gene expression analysis. In doing so, we were able to systematically evaluate the sensitivity, precision, and accuracy of various approaches to single-cell RNA-seq. Our results show that it is possible to use single-cell RNA-seq to perform quantitative transcriptome measurements of individual cells, that it is possible to obtain quantitative and accurate gene expression measurements with a relatively small number of sequencing reads, and that when such measurements are performed on large numbers of cells, one can recapitulate the bulk transcriptome complexity, and the distributions of gene expression levels found by single-cell qPCR. 109 single-cell human transcriptomes were analyzed in total; 96 using nanoliter volume sample processing on a microfluidic platform, Nextera library prep (biological replicates); 3 using the SMARTer cDNA synthesis kit, Nextera library prep (biological replicates); 3 using the Transplex cDNA synthesis kit, Nextera library prep (biological replicates); 7 using the Ovation Nugen cDNA synthesis kit (biological replicates) where 3 used Nextera library prep and 4 used NEBNext library prep. In addition, 4 bulk RNA samples were sequenced: bulk RNA generated using ~1 million pooled cells was used to make bulk libraries, 2 of which were made using SMARTer cDNA synthesis kit (technical replicates) and 2 made using Superscript RT kit with no amplification (technical replicates). All 4 bulk samples were made into libraries using Nextera.
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