NextSeq 500 paired end sequencing; GSM2898321: HIV_s1; Homo sapiens; RNA-Seq

HIV_s1

Human immunity relies on the coordinated responses of many cellular subsets and functional states. Inter-individual variations in cellular composition and communication could thus potentially alter host protection. Here, we explore this hypothesis by applying single-cell RNA-Seq to examine viral responses among the dendritic cells (DCs) of three elite controllers (ECs) of HIV-1 infection. We discover a highly functional antiviral DC state in ECs whose fractional abundance after in vitro exposure to HIV-1 correlates with higher CD4+ T cell counts and lower HIV-1 viral loads, and that effectively primes polyfunctional T cell responses in vitro. We identify and validate select immunomodulators that increase the fractional abundance of this state in primary peripheral blood mononuclear cells (PBMCs) from healthy individuals in vitro. Overall design: Single-cell RNA-seq profiling of HIV-1-exposed cDCs and media controls from 3 elite controllers used to identify reproducible gene expression programs associated with cell-intrinsic HIV-1 immune recognition.

A Reproducibility-Based Computational Framework Identifies An Inducible, Enhanced Antiviral Dendritic Cell State In HIV-1 Elite Controllers (scRNA-Seq)

Submitted by Gene Expression Omnibus on 02-AUG-2016

Single cells lysates were put directly into SPRI [Trombetta et al CPMB 2014] [Single cell Whole Transcriptome Amplification]: Following sorting, 96-well plates of single cells were whole transcriptome amplified as described in Trombetta et al. Lysed cell samples were cleaned with 2.2x volume AMPure XP SPRI beads (Beckman Coulter). Reverse transcription and PCR were then performed on the samples.  For population samples total RNA was isolated using a column (RNeasy plus Micro RNA kit; Qiagen) following manufacturer's instructions. Two μL of extracted RNA were added to 8 μL of water and cleaned with 2.2x volume beads. While transcriptome amplification wa performed on these samples analogous to the amplification of the single cell samples. [Preparation of cDNA Libraries  for RNAseq]: WTA products were diluted to a concentration of 0.1 to 0.4 ng/μL and tagmented and amplified using Nextera XT DNA Sample preparation reagents (Illumina). Tagmentation was performed according to manufacturer's instructions, modified to use ¼ the recommended volume of reagents, extended tagmentation time to 10 minutes and extended PCR time to 60s. PCR primers were ordered form Integrated DNA Technologies. Nextera products were then cleaned twice with at 0.9x volume of SPRI beads and eluted in water. The library was quantified using Qubit and analyzed using a high sensitivity DNA chip. The library was diluted to 2.2 pM and sequenced on a NextSeq 500 (Illumina) Indexing length: 8bp

A Reproducibility-Based Computational Framework Identifies An Inducible, Enhanced Antiviral Dendritic Cell State In HIV-1 Elite Controllers (scRNA-Seq)

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