Illumina HiSeq 2500 paired end sequencing; Impact of aging on cell-to-cell transcriptional variability during immune stimulation
Impact of aging on cell-to-cell transcriptional variability during immune stimulation
Cells were purified from spleen in two different sub-species of mice (C57BL/6 and CAST/EiJ; all male animals) using EASY cell strainer (30 μm, Greiner BioOne), cell separation media (lympholyte, #CL5035) and the CD4+ CD62L+ T Cell Isolation Kit II (Miltenyi Biotec, #130-093-227). We use a microfluidics-based approach (5–10 μm Auto Prep Integrated Fluidic Circuit - IFC, Fluidigm, San Francisco, CA) to capture single cells and the C1 Single cell Auto Prep System (Fluidigm) to prepare single-cell RNA-Seq libraries CD4+ T cells. Purified cells were directly loaded into the microfluidics (naive CD4+ T cells) or were stimulated on 96 round-plates coated with anti-CD3ε and anti-CD28 for 3 hours (activated CD4+ T cells).  Naive and effector memory (EM) CD4+ T cells were purified from spleens of both young and old C57/BL6 mice by FACS.  Briefly, spleens were harvested from both young and old animals and single cell suspensions were obtained by meshing through a cell strainer (70 μm). B cells were depleted from cell suspensions by MACS using CD19 microbeads (Miltenyi Biotec, #130-052-201) and red blood cells were lysed with RBC lysis buffer (Biolegend, #B205551). The enriched cell fraction was then stained with Fixable eFluor 780 viability dye (eBioscience) following by Fc receptor blocking with TruStain fcXTM (clone: 39, Biolegend) and subsequent staining with a panel of fluorescence-conjugated antibodies against CD4 (clone: RM4-5, BioLegend), CD44 (clone: IM7, BioLegend), CD62L (clone: MEL-14, BioLegend), CD24 (clone: M1/69, BioLegend), Qa2 (clone: 695H1-9-9, BioLegend), CD69 (clone: H1.2F3, BioLegend) and PD-1 (clone: RMP1-30, BioLegend).  Stained cells were immediately sorted using a 5-laser Aria IIu SORP instrument (BD Biosciences). Lymphocytes were gated by the use of forward scatter (FSC-A) and side scatter (SSC-A). Cell doublets were excluded according to area and height of forward scatter (FSC-A/FSC-H). Dead cells were removed using viability dye. PD-1+ CD4+ T cells were excluded and PD-1-ve CD4+ T cells were t further separated into naive and EM CD4+ T cell subsets according to their CD44 and CD62L expression. Cells with a mature CD24lo Qa2hi phenotype were then gated from naive and EM subsets and CD69+ cells were removed. Purified cells were directly loaded into the microfluidics (naive CD4+ T cells) or were stimulated on 96 round-plates coated with anti-CD3 and anti-CD28 for 3 hours (activated CD4+ T cells) cDNAs were made on-chip (Fluidigm C1, 5-10um IFC) with Clontech SMARTer Ultra Low RNA kit. ERCC spike-in RNA (Ambion) (1 µL diluted at 1:50,000) was added to the C1 lysis mix  Sequencing libraries were prepared using Illumina Nextera XT kit (Illumina) using the recommendation in the Fluidigm protocol (# PN 100-7168 K1).
Experimental Factor: do11648
single cell ID
Experimental Factor: Mus musculus domesticus
organism
Experimental Factor: 17.4
age
Experimental Factor: anti-CD3e and anti-CD28 for 3 hours
stimulus