inDrop

inDrop

inDrop is used for high-throughput single-cell labeling. This approach is similar to Drop-seq, but it uses hydrogel microspheres to introduce the oligonucleotides.

Single cells from a cell suspension are isolated into droplets containing lysis buffer. After cell lysis, cell droplets are fused with a hydrogel microsphere containing cell-specific barcodes and another droplet with enzymes for RT. 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 pooled and 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, Quartz-Seq, Hi-SCL.

Pros:

• High throughput single-cell transcriptome profiling using microfluidics system
• Highly scalable to larger cell quantities
• No fragmentation step

Cons:

• Low mRNA capture efficiency of ~7%
• Droplets may contain two cells or two different types of barcodes

• inDrop: Klein A. M., Mazutis L., Akartuna I., Tallapragada N., Veres A., et al. (2015) Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 161: 1187-1201

1. Bian Q. and Cahan P. Computational Tools for Stem Cell Biology. Trends Biotechnol. 2016;
2. Zhao Q.-Y., Gratten J., Restuadi R. and Li X. Mapping and differential expression analysis from short-read RNA-Seq data in model organisms. Quantitative Biology. 2016;4:22-35
3. Grun D. and van Oudenaarden A. Design and Analysis of Single-Cell Sequencing Experiments. Cell. 2015;163:799-810
4. Saadatpour A., Lai S., Guo G. and Yuan G. C. Single-Cell Analysis in Cancer Genomics. Trends Genet. 2015;31:576-586

1. Derr A., Yang C., Zilionis R., et al. End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data. Genome Res. 2016;26:1397-1410
2. Klein A. M., Mazutis L., Akartuna I., Tallapragada N., Veres A., et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell. 2015;161:1187-1201