Category: High-Definition Biotechnology

1137-C - Miniaturized Full Length Single Cell RNA sequencing Protocol Utilizing Acoustic Dispensing and SMART-Seq v4 Kit

Tuesday, February 6, 2018
2:00 PM - 3:00 PM

The rapidly growing field of single cell analytics seeks to answer questions about cellular diversity in various diseases and developmental processes and can lead to the discovery of novel cell subtypes and stages. The genetic profiling of single cells of cancerous tissue will open up new views to their possible heterogeneity, which might have a significant effect on the treatment of an individual patient.

Higher cell throughputs for single cell sequencing are needed for identification of rare cell types which can lead to better understanding of the cellular heterogeneity. Acoustic dispensing is a powerful method for high precision dosing of assay reagents in very small volumes and is thus ideal also for miniaturization of full length single cell mRNA sequencing reactions. These reagents are expensive, so smaller reaction volumes bring significant savings in reagent costs. In addition acoustic dispensing is touchless reducing the risk of contamination.

Here we describe how we have utilized miniaturized protocol of SMART-Seq v4 Ultra Low Input RNA Kit (Takara Bio) and Nextera XT DNA Library Prep Kit (Illumina) dispensed acoustically to analyze full length RNA transcripts from differentiated induced pluripotent stem cells (iPS cells). Libraries from each cell were sequenced with Illumina HiSeq 2500 using paired-end sequencing and 101 read length. We have used approximately ΒΌ of standard reaction volumes. The alignment of the processed paired-end reads was performed against the human reference genome (Ensembl GRCh38) using the gap-aware STAR aligner. On average 560 000 paired-end read-pairs were generated per cell. Scaling down the SMART-Seq v4 protocol enables better cost efficacy for full length RNA transcript analysis and can be used for analysis of various cell types.

Laura Turunen

Laboratory Coordinator
FIMM /High Throughput Biomedic
Helsinki University, Uusimaa, Finland

Laura Turunen has worked at FIMM High Throughput Biomedicine (HTB) team for eight years as automation specialist and compound manager. She's responsible for the various automated equipment, including various liquid handlers and dispensers, and the compound collections consisting approximately 130 000 compounds from commercial sources. She's also planning and running various projects for users of the HTB lab services.