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. 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.

Global delay in nascent strand DNA methylation

“…Library generation and analysis was done as described in Ref 37 . Briefly, RNA-seq reads were first trimmed using Trimmomatic 38 .…”

Circulating CXCR5+CXCR3+PD-1lo Tfh-like cells in HIV-1 controllers with neutralizing antibody breadth

“…Subsequently, total RNA was extracted using a commercial kit recommended for low numbers of cells (mirVana Isolation Kit, Life Technologies). RNA sequencing (RNA-Seq) libraries from mDCs from each patient group were generated as previously described (57). Briefly, whole transcriptome amplification (WTA) and tagmentation-based library preparation were performed using SMART-seq2 (57), followed by sequencing on a NextSeq 500 instrument (Illumina).…”

DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs

“…RNA was isolated as described above. Smart-Seq2 libraries were generated and sequenced using a Broad Genomics Platform (86)(87)(88). The quality of the sequencing files was initially assessed with the FastQC tool.…”