Collection Citation: John J. Trombetta, David Gennert, Diana Lu, Rahul Satija, Alex K. Shalek, Aviv Regev 2018. Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.pbhdij6
Manuscript citation:
Trombetta J. (2014). Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing. Curr Protoc Mol Biol. ; 107: 4.22.1–4.22.17. doi:10.1002/0471142727.mb0422s107
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: April 08, 2018
Last Modified: July 13, 2018
Collection Integer ID: 11337
Keywords: SMART-Seq, Single-Cell RNA-Seq, Transcriptomics, cell type, Single-cell, low input RNA-Seq, SMART-Seq2, Next-generation sequencing, template-switching, seq libraries for next generation sequencing, transcriptomic, reverse transcriptase, next generation sequencing, cell rna, mrna transcript, field of transcriptomic, complementary dna, length mrna transcript, sequencing, seq libraries for next generation, cdna, seq library, universal priming sequence, common sequence, individual cell, dna, significant differences between individual cell, seq
Abstract
For the past several decades, due to technical limitations, the field of transcriptomics has focused on population-level measurements that can mask significant differences between individual cells. With the advent of single-cell RNA-Seq, it is now possible to profile the responses of individual cells at unprecedented depth and thereby uncover, transcriptome-wide, the heterogeneity that exists within these populations. Here, we describe a method that merges several important technologies to produce, in high-throughput, single-cell RNA-Seq libraries. Complementary DNA (cDNA) is made from full-length mRNA transcripts using a reverse transcriptase that has terminal transferase activity. This, when combined with a second “template-switch” primer, allows for cDNAs to be constructed that have two universal priming sequences. Following preamplification from these common sequences, Nextera XT is used to prepare a pool of 96 uniquely indexed samples ready for Illumina sequencing.
