Massively parallel nanowell-based single-cell gene expression profiling.

BACKGROUND: Technological advances have enabled transcriptome characterization of cell types at the single-cell level providing new biological insights. New methods that enable simple yet high-throughput single-cell expression profiling are highly desirable. RESULTS: Here we report a novel nanowell-based single-cell RNA sequencing system, ICELL8, which enables processing of thousands of cells per sample. The system employs a 5,184-nanowell-containing microchip to capture ~1,300 single cells and process them. Each nanowell contains preprinted oligonucleotides encoding poly-d(T), a unique well barcode, and a unique molecular identifier. The ICELL8 system uses imaging software to identify nanowells containing viable single cells and only wells with single cells are processed into sequencing libraries. Here, we report the performance and utility of ICELL8 using samples of increasing complexity from cultured cells to mouse solid tissue samples. Our assessment of the system to discriminate between mixed human and mouse cells showed that ICELL8 has a low cell multiplet rate (< 3%) and low cross-cell contamination. We characterized single-cell transcriptomes of more than a thousand cultured human and mouse cells as well as 468 mouse pancreatic islets cells. We were able to identify distinct cell types in pancreatic islets, including alpha, beta, delta and gamma cells. CONCLUSIONS: Overall, ICELL8 provides efficient and cost-effective single-cell expression profiling of thousands of cells, allowing researchers to decipher single-cell transcriptomes within complex biological samples.

Target enrichment using parallel nanoliter quantitative PCR amplification.

BACKGROUND: Next generation targeted resequencing is replacing Sanger sequencing at high pace in routine genetic diagnosis. The need for well validated, high quality enrichment platforms to complement the bench-top next generation sequencing devices is high. RESULTS: We used the WaferGen Smartchip platform to perform highly parallelized PCR based target enrichment for a set of known cancer genes in a well characterized set of cancer cell lines from the NCI60 panel. Optimization of PCR assay design and cycling conditions resulted in a high enrichment efficiency. We provide proof of a high mutation rediscovery rate and have included technical replicates to enable SNP calling validation demonstrating the high reproducibility of our enrichment platform. CONCLUSIONS: Here we present our custom developed quantitative PCR based target enrichment platform. Using highly parallel nanoliter singleplex PCR reactions makes this a flexible and efficient platform. The high mutation validation rate shows this platform's promise as a targeted resequencing method for multi-gene routine sequencing diagnostics.

STRT-seq-2i: dual-index 5' single cell and nucleus RNA-seq on an addressable microwell array.

Single-cell RNA-seq has become routine for discovering cell types and revealing cellular diversity, but archived human brain samples still pose a challenge to current high-throughput platforms. We present STRT-seq-2i, an addressable 9600-microwell array platform, combining sampling by limiting dilution or FACS, with imaging and high throughput at competitive cost. We applied the platform to fresh single mouse cortical cells and to frozen post-mortem human cortical nuclei, matching the performance of a previous lower-throughput platform while retaining a high degree of flexibility, potentially also for other high-throughput applications.

Comparison of on-chip and off-chip microfluidic kinase assay formats.

Kinases represent an important class of targets for pharmaceutical drug development. Microfluidic devices capable of running kinase assays with either an on-chip or an off-chip enzymatic reaction have been developed. For the on-chip assay, reagent addition, mixing, enzymatic reaction, and electrophoretic separation and detection of substrate and product all take place in the channels of the microfluidic chip. For the off-chip assay, the reaction takes place in a microtiter plate, whereas the electrophoretic separation and detection of substrate and product take place in the channels of the chip. To probe differences between the on-chip and off-chip assays, a panel of commercially available kinase inhibitors was assayed at 10 microM against cyclic AMP-dependent protein kinase A, glycogen synthase kinase 3beta, mitogen- and stress-activated protein kinase, and Akt1 using both the off-chip and on-chip assays. Good correlation was observed between inhibition measured by the two methods, with most of the differences in measured inhibition being attributed to compound solubility and enzyme concentration effects. Microfluidic devices represent an attractive platform for kinase assays due to high data quality and the possibility of on-chip assay integration, leading to reagent and labor savings.