Hi-SCL
High-Throughput Single-Cell Labeling
Hi-SCL generates transcriptome profiles for thousands of single cells using a custom microfluidics system, similar to Drop-Seq and inDrop (Rotem et al., 2015).
Single cells from cell suspension are isolated into droplets containing lysis buffer. After cell lysis, cell droplets are fused with a droplet containing cell-specific barcodes and another droplet with enzymes for RT. The droplets from all the wells are pooled and subjected to isothermal reactions for RT. The barcodes anneal to poly(A)+ mRNAs and act as primers for reverse transcriptase. Now that each mRNA strand has cell-specific barcodes, the droplets are broken, and the cDNA is purified. The 3′ ends of the cDNA strands are ligated to adapters, amplified, annealed to indexed primers, and amplified further before sequencing.
Similar methods: CEL-Seq, Drop-seq, MARS-Seq, CytoSeq, inDrop, Quartz-Seq
Advantages:
- High-throughput, single-cell transcriptome profiling using a microfluidics system
- Low cost: $0.1 per cell (for experiment with 100 cells)
- Highly scalable to larger cell quantities
- No fragmentation step
Disadvantages:
- Lack of UMI in oligonucleotides may create amplification noise
- Droplets may contain 2 cells or 2 different types of barcodes
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Mato Prado M., Frampton A. E., Stebbing J. and Krell J. Single-cell sequencing in cancer research. Expert Rev Mol Diagn. 2016;16:1-5
References:
Rotem A., Ram O., Shoresh N., et al. High-Throughput Single-Cell Labeling (Hi-SCL) for RNA-Seq Using Drop-Based Microfluidics. PLoS One. 2015;10:e0116328
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History: Hi-SCL
Revision by sbrumpton on 2017-06-21 07:50:22 - Show/Hide
High-Throughput Single-Cell Labeling
Hi-SCL generates transcriptome profiles for thousands of single cells using a custom microfluidics system, similar to Drop-Seq and inDrop (Rotem et al., 2015).
Single cells from cell suspension are isolated into droplets containing lysis buffer. After cell lysis, cell droplets are fused with a droplet containing cell-specific barcodes and another droplet with enzymes for RT. The droplets from all the wells are pooled and subjected to isothermal reactions for RT. The barcodes anneal to poly(A)+ mRNAs and act as primers for reverse transcriptase. Now that each mRNA strand has cell-specific barcodes, the droplets are broken, and the cDNA is purified. The 3' ends of the cDNA strands are ligated to adapters, amplified, annealed to indexed primers, and amplified further before sequencing.
Similar methods: CEL-Seq, Drop-seq, MARS-Seq, CytoSeq, inDrop, Quartz-Seq
Advantages:- High-throughput, single-cell transcriptome profiling using a microfluidics system
- Low cost: $0.1 per cell (for experiment with 100 cells)
- Highly scalable to larger cell quantities
- No fragmentation step
Disadvantages:- Lack of UMI in oligonucleotides may create amplification noise
- Droplets may contain 2 cells or 2 different types of barcodes
Reagents:Illumina Library prep and Array Kit SelectorReviews:Mato Prado M., Frampton A. E., Stebbing J. and Krell J. Single-cell sequencing in cancer research. Expert Rev Mol Diagn. 2016;16:1-5References:Rotem A., Ram O., Shoresh N., et al. High-Throughput Single-Cell Labeling (Hi-SCL) for RNA-Seq Using Drop-Based Microfluidics. PLoS One. 2015;10:e0116328