FRISCR

FRISCR

FRISCR characterizes transcriptome profiles from fixed and stained single cells. The method uses a combination of molecular barcodes and Tn5 tagmentation to identify each cDNA fragment uniquely from every cell.

The cell suspension is fixed with paraformaldehyde, permeabilized, and immunostained. Individual cells are sorted into tubes by FACS. These cells are lysed and reverse-crosslinked by incubation at 56°C for 1 hour. mRNA from the cells is isolated by dT25 magnetic bead pull-down. The mRNA sequencing library is prepared according to the Smart-seq2 procedure: 1) template-switching RT with Moloney murine leukemia virus reverse transcriptase; 2) the resulting cDNAs are PCR amplified; and 3) the cDNA library is generated using the Nextera XT Library Preparation Kit. The cDNA fragments are flanked with adapters and are ready for sequencing.

Pros:

• Full-length mRNA transcriptome profiling from fixed and stained single-cells
• Immunostaining enables targeting of rare cell populations
• Generate full-length mRNA reads
• Significantly more mRNA recovered compared to fixed cells from Triton-X100 lysis

Cons:

• 3' to 5' bias

• FRISCR: Thomsen E. R., Mich J. K., Yao Z., Hodge R. D., Doyle A. M., et al. (2016) Fixed single-cell transcriptomic characterization of human radial glial diversity. Nat Methods 13: 87-93

1. Wen L. and Tang F. Single-cell sequencing in stem cell biology. Genome Biol. 2016;17:71

Thomsen E. R., Mich J. K., Yao Z., Hodge R. D., Doyle A. M., et al. (2016) Fixed single-cell transcriptomic characterization of human radial glial diversity. Nat Methods 13: 87-93