title: Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing.
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.
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts.
authors.forename: John J
authors.initials: JJ
authors.lastname: Trombetta
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts.
authors.forename: David
authors.initials: D
authors.lastname: Gennert
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts.
authors.forename: Diana
authors.initials: D
authors.lastname: Lu
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts.
authors.forename: Rahul
authors.initials: R
authors.lastname: Satija
authors.affiliation: Department of Chemistry and Chemical Biology and Department of Physics, Harvard University, Cambridge, Massachusetts.
authors.forename: Alex K
authors.initials: AK
authors.lastname: Shalek
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts.|Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Aviv
authors.initials: A
authors.lastname: Regev
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger
keywords: Next-generation sequencing; SMART-Seq; SMART-Seq2; cell type; low-input RNA-Seq; single cell; single-cell RNA-Seq; template-switching; transcriptomics