A Sensitive and Integrated Approach to Profile Messenger RNA from Samples with Low Cell Numbers

Abstract

Transcriptomic profiling by RNA sequencing (RNA-Seq) represents the preferred approach to measure genome-wide gene expression for understanding cellular function, tissue development, disease pathogenesis, as well as to identify potential biomarkers and therapeutic targets. For samples with small cell numbers, multiple methods have been described to increase the efficiency of library preparation and to reduce hands-on time and costs. This chapter reviews our approach, which combines flow cytometry and the most recent high-resolution techniques to perform RNA-Seq for samples with low cell numbers as well as for single-cell samples. Our approach reduces technical variability while increasing sensitivity and efficiency. Thus, it is well-suited for large-scale gene expression profiling studies with limited samples for basic and clinical studies.

Cutting Edge: Transcriptional Profiling Reveals Multifunctional and Cytotoxic Antiviral Responses of Zika Virus–Specific CD8+ T Cells

“…The patients' CD8 + T cells that did not produce IFN-g in response to epitope stimulation from these same cultures as well as total unstimulated CD8 + T cells were also collected for comparison with their ZIKV-specific IFN-g-producing CD8 + T cells. After sorting, microscaled RNA sequence determinations were performed on low cell numbers (12). We then analyzed the global gene expression patterns using PCA of the 500 most variable genes obtained after RNA sequencing (Fig.…”

“…Two hundred cells for each condition were collected. To generate full-length transcriptomes from the low cells per sample, we adapted the Smart-Seq2 protocol (12). Bioinformatics analysis to obtain the raw counts and differentially expressed genes were performed as previously described (13).…”

Dengue-specific CD8+ T cell subsets display specialized transcriptomic and TCR profiles

“…Right after sorting, tubes were vortexed at medium speed, spun for 5 minutes at more than 2000 g, and stored at -80°C until the completion of the whole set of samples. Four microliters of each sample was amplified following the Smart-seq2 protocol (55,56). Briefly, mRNA was captured using poly-dT oligonucleotides and directly reverse-transcribed into full-length cDNA using the described template-switching oligonucleotide (55,56).…”

“…Four microliters of each sample was amplified following the Smart-seq2 protocol (55,56). Briefly, mRNA was captured using poly-dT oligonucleotides and directly reverse-transcribed into full-length cDNA using the described template-switching oligonucleotide (55,56). cDNA was amplified by PCR for 18 cycles and purified using AMPure XP magnetic beads (0.9:1 [vol/vol] ratio; Beckman Coulter).…”

Impact of Genetic Polymorphisms on Human Immune Cell Gene Expression

“…S6F. RNA isolation and preparation of RNA-Seq libraries was done following the low-input protocol described previously (Rosales et al, 2018). Differentially expressed (DE) genes across samples from treatment with siRNA pool for FAM13A or non-targeting siRNA were identified by pairwise comparisons of all samples including replicates, using DESeq2 (Love et al, 2014); we considered a transcript to be expressed differentially in a comparison when the DESeq2 analysis resulted in a Benjamini-Hochberg adj.…”