InDrop

High-Throughput Single-Cell Labeling with Indexing Droplets

inDrop is used for high-throughput single-cell labeling (Klein et al., 2015). 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

Advantages:

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

Disadvantages:

  • Low mRNA capture efficiency of ~7%
  • Droplets may contain 2 cells or 2 different types of barcodes


Reagents:

Illumina Library prep and Array Kit Selector



Reviews:

Bian Q. and Cahan P. Computational Tools for Stem Cell Biology. Trends Biotechnol. 2016;

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

Grun D. and van Oudenaarden A. Design and Analysis of Single-Cell Sequencing Experiments. Cell. 2015;163:799-810

Saadatpour A., Lai S., Guo G. and Yuan G. C. Single-Cell Analysis in Cancer Genomics. Trends Genet. 2015;31:576-586



References:

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