mcSCRB-seq: sensitive and powerful single-cell RNA sequencing

Abstract

SummarySingle-cell RNA sequencing (scRNA-seq) has emerged as the central genome-wide method to characterize cellular identities and processes. While performance of scRNA-seq methods is improving, an optimum in terms of sensitivity, cost-efficiency and flexibility has not yet been reached. Among the flexible plate-based methods "Single-Cell RNA-Barcoding and Sequencing" (SCRB-seq) is one of the most sensitive and efficient ones. Based on this protocol, we systematically evaluated experimental conditions such as reverse transcriptases, reaction enhancers and PCR polymerases. We find that adding polyethylene glycol considerably increases sensitivity by enhancing cDNA synthesis. Furthermore, using Terra polymerase increases efficiency due to a more even cDNA amplification that requires less sequencing of libraries. We combined these and other improvements to a new scRNA-seq library protocol we call "molecular crowding SCRB-seq" (mcSCRB-seq), which we show to be the most sensitive and one of the most efficient and flexible scRNA-seq methods to date.

Tutorial: guidelines for the experimental design of single-cell RNA sequencing studies

“…The SCRB-seq 50 protocol incorporates single-cell barcodes and UMI in the polyT-primer, enabling 3' amplification of transcripts, and like STRT-seq, early indexing allows cell pooling to reduce costs. The RNA capture efficiency of the original protocol was improved by increasing the RT mix density: molecular crowding SCRB-seq (mcSCRB-seq 51 ) includes polyethylene glycol to increase binding event probabilities. In addition, the PCR enzyme was switched from KAPA to the Terra polymerase to further improve library complexity.…”

“…Most importantly, SuperScript II carries point mutations that inactivate its RNAse H domain, impairing competitive RNA degradation during cDNA synthesis. Alternative RT enzymes have been reported, with similar or superior performances, such as Maxima H (used in SCRB-seq 50,51 ) or SMARTscribe in the SMARter v4 kit (Takara Bio). Protocols that do not require template switching and generate second strands by other means, such as polyAtailing or random priming 130,131 , can use SuperScript III, which carries different point mutations in the RNA polymerase, and displays increased thermal stability.…”

Quantitative single-cell transcriptomics

“…So more flexible, lower-throughput, well-based methods and efficient, high-throughput, droplet-based methods will likely coexist to accommodate different needs. Moreover, new methods continue to be developed at impressive speeds ( Supplementary Table S1 ) [ 75 ]. For example, a recent preprint describes a setup that deposits cells or nuclei by FACS or limiting dilution in thousands of micro-wells and also allows imaging of cells [ 70 ], representing a good compromise of flexibility and throughput.…”

“…For all protocols, the crucial step is efficiently converting RNA into cDNA, which depends on a combination of enzyme properties, buffer conditions, volume and RNA degradation rates. Systematic optimizations have improved the sensitivity for several protocols [ 75–78 ]. The most sensitive ones reach conversion efficiencies of almost 50% [ 75 ] and can probably still be improved given the complex interaction of many factors [ 75 ].…”

zUMIs - A fast and flexible pipeline to process RNA sequencing data with UMIs

“…Next, single-cell RNA-seq libraries were constructed from one 96-well plate using a slightly modified version of the mcSCRB-seq protocol. Reverse transcription was performed as described previously [ 40 ], with the only change being the use of KAPA HiFi HotStart enzyme for PCR amplification of cDNA. Resulting libraries were sequenced using an Illumina HiSeq1500 with 16 cycles in Read 1 to decode cell BCs (6 bases) and UMIs (10 bases) and 50 cycles in Read 2 to sequence into the cDNA fragment, obtaining ∼227 million reads.…”