High-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. Researchers from the RIKEN Institute thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Their improvements in several of the reaction steps of Quartz-Seq2 allow the researchers to effectively convert initial reads to UMI counts (at a rate of 30%-50%). To demonstrate the power of Quartz-Seq2, they analyzed transcriptomes from a cell population of in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of sequence reads.
Overview of Quartz-Seq2 experimental processes
Quartz-Seq2 consisted of five steps. (1) Each single cell in a droplet was sorted into lysis buffer in each well of a 384-well PCR plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well was reverse-transcribed into first-strand cDNA with reverse transcription primer, which had a unique cell barcode. We prepared 384 or 1,536 kinds of cell barcode (CB) with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). (4) Collected first-strand cDNAs were purified and concentrated for subsequent whole-transcript amplification. In the poly-A tailing step, purified cDNA was extended with a poly-A tail by terminal deoxynucleotidyl transferase (TdT). (5) For conversion from amplified cDNA to sequence library DNA, we fragmented the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA was ligated with a truncated Y-shaped sequence adaptor, which had an Illumina flow-cell binding sequence (P7) and a pool barcode sequence (PB).