Posted by Anna Williams on August 17, 2018:

https://www.ncbi.nlm.nih.gov/pubmed/?term=28818938

Comprehensive single-cell transcriptional profiling of a multicellular organism (PDF version – Please do not further distribute the PDF until the paper has become freely available from the journal)

This is a methods paper describing single-cell combinatorial indexing RNA sequencing (sci-RNA-seq), a new high throughput single cell RNA-seq method which does not require the isolation of a single cell within a physical compartment. In this method, tissue is dissociated to single cells, then fixed and permeabilized with methanol and plated on a 96 well plate with 1,000-10,000 cells per well. The in situ reverse transcriptase is performed with the first barcode, which add a unique identifier to transcripts from all cells within that well. The cells are then mixed together and sorted with FACs, dublets are removed (more than one cell clumped together), and the cells are distributed on a 96 well plate with 10-100 cells per well. Then they perform second strand synthesis, transposition, lysis, and PCR amplification with a second barcode. The combination of the first and second barcode give a unique identifier for each individual cell. The transcripts are pooled and sequenced.