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Curr Protoc Mol Biol DOI:10.1002/0471142727.mb0422s107

Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing.

Publication TypeJournal Article
Year of Publication2014
AuthorsTrombetta, JJ, Gennert, D, Lu, D, Satija, R, Shalek, AK, Regev, A
JournalCurr Protoc Mol Biol
Volume107
Pages4.22.1-17
Date Published2014 Jul 01
ISSN1934-3647
KeywordsAnimals, DNA, Complementary, Gene Library, High-Throughput Nucleotide Sequencing, Humans, RNA, Messenger, Sequence Analysis, RNA
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. This unit describes 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.

URLhttp://dx.doi.org/10.1002/0471142727.mb0422s107
DOI10.1002/0471142727.mb0422s107
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/24984854?dopt=Abstract

Alternate JournalCurr Protoc Mol Biol
PubMed ID24984854
PubMed Central IDPMC4338574
Grant ListF32 HD075541 / HD / NICHD NIH HHS / United States
1F32HD075541-01 / HD / NICHD NIH HHS / United States
DP1OD003958-01 / OD / NIH HHS / United States
P50 HG006193 / HG / NHGRI NIH HHS / United States
/ / Howard Hughes Medical Institute / United States
1P50HG006193-01 / HG / NHGRI NIH HHS / United States
DP1 OD003958 / OD / NIH HHS / United States