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URL: https://dx.doi.org/DOI:10.17504/protocols.io.s4hegt6

Authors: Jeremy Worley*, Hongxu Ding*, Erin Bush, Peter Sims, and Andrea Califano

Summary: Since 2012, numerous single-cell RNA sequencing (scRNA-seq) technologies have been introduced. Most involve either significant startup cost, an expert operator, or expensive library preparation. We developed a plate-based library prep that incorporates pre-amplification pooling, unique molecular identifiers (UMIs), and mRNA enrichment using silanol coated magnetic beads. Our method, which builds off of Bush et al. (Nature Communications, 2017) and Picelli et al. (Nature Protocols, 2014), uses readily available equipment, is easy to automate, and costs a fraction of commercial kits. In addition, our method has been optimized to reduce barcode cross contamination that can occur in pooled pre-amplification reactions. This method allows for plate-based 3’ scRNA-seq with UMIs at relatively low cost, using standard lab equipment.NOTE (2018/08/29): we are currently testing optimizations described by Bagnoli et al. (Nature Communications, 2018). This protocol may be updated accordingly. 

Affiliations: Columbia University, Columbia University, Columbia University, Columbia University, Columbia University

Version: 1

Publication Date: 2018

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