ScRNA-seq of mESC and mouse CD4+ cells using different buffers and enzymes

ABSTRACT: Existing protocols for full-length single-cell RNA sequencing (scRNA-seq) produce libraries of high complexity (thousands of distinct genes) with outstanding sensitivity and specificity of transcript quantification. These full-length libraries have the advantage of allowing probing of transcript isoforms, are informative regarding single nucleotide polymorphisms, and allow assembly of the VDJ region of the T- and B-cell receptor sequences. Since full length protocols are mostly plate-based at present, they are also suited to profiling cell types where cell numbers are limiting, such as rare cell types during development for instance. A disadvantage of these methods has been the scalability and cost of the experiments, which has limited their popularity as compared to droplet-based and nanowell approaches. Here, we describe an automated protocol for full-length scRNA-seq, including both an in-house automated SMART-seq2 protocol, and a commercial kit-based workflow. We discuss these two protocols in terms of ease-of-use, equipment requirements, running time, cost per sample and sequencing quality. By benchmarking the lysis buffers, reverse transcription enzymes and their combinations, we propose an optimized in-house automated protocol with dramatically reduced cost. These pipelines have been employed successfully for several research projects allied with the Human Cell Atlas initiative (www.humancellatlas.org) and are available on protocols.io.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Mus musculus

SUBMITTER: Zhichao Miao 

PROVIDER: E-MTAB-9345 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress