GSM2844832: C57BL/6J_Male_RestingBcell_173; Mus musculus; RNA-Seq
Naïve CD4+ T lymphocytes were purified from single-cell suspensions by MACS depletion of non-CD4+ T cells as well as regulatory T cells and TCRγ/δ+ T cells, using a cocktail of specific biotin conjugated antibodies against CD8a (Ly-2), CD45R (B220), CD49b (DX5), CD11b (Mac-1), Ter-119, CD25 and TCRγ/δ+ using the CD4+ CD62L+ T Cell Isolation Kit II (Miltenyi) to obtain an unlabeled pre-enriched CD4+ T cell fraction. Collected cells were suspended in PBS containing 2% FBS and Fc receptors were blocked with FcR blocking reagent for 10 minutes at 4°C (Miltenyi). Cells were further fluorescently stained with a combination of CD4-FITC (Miltenyi), CD44-APC clone IM7 (eBioscience), CD62L clone MEL-14 (BD Pharmingen), eFluor® 450 CD25 clone PC61.5 (eBioscience), for 10 minutes at 4°C, washed and then stained with DAPI (Life Technologies) to cell sort the live population of naïve CD4+, CD62Lhi, CD44lo, CD25- T lymphocytes using the MoFlo XDP system (Beckman Coulter). Samples were gated on single cells for doublet discrimination and non-viable cells were excluded using the viability dye 4', 6 – diamidine – 2 – phenylindole (DAPI). AllPrep DNA/RNA Mini Kit (Qiagen) was used to perform simultaneous isolation of RNA and DNA from 10^6 sorted naïve CD4+ T cells and resting B cells.  All RNA samples were subjected to in-column RNase-Free DNase treatment (Qiagen) to remove traces of DNA contamination. RNA integrity was subsequently assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies) by using the RNA 6000 Pico Kit (Agilent Technologies). For each sample type 3 biological replicates were generated from male and female samples. Purified male quiescent naïve CD4+ T and resting B cells were loaded onto a 5-10 μm C1 Single-Cell Auto Prep Integrated Fluidic Circuits (IFC) and captured following manufactures' instructions. Capture efficiency for each cell type and IFC was determined using a microscope in order to remove in further analysis samples with no cell capture, debris or with two or more cells captured. Synthetic ERCC ExFold RNA Spike-In Mixes (Ambion®) were added at a 1:5000 (B cells) and 1:1000 (T cells) dilution to the cell lysis mix as controls to estimate the level of technical noise in further analysis. Cell lysis, reverse transcription and cDNA pre-amplification was performed in the Fluidigm C1™ Single-Cell Auto Prep System using the C1 Single-Cell Auto Prep Array (IFCs), the Auto Prep Reagent Kit for mRNA Seq (Fluidigm) and the SMARTer® Ultra™ Low RNA Kit (Clontech). Universal amplification was performed utilising the Advantage® 2 PCR Kit (Clontech). cDNA was harvested, quantified by Qubit® assays and diluted to 0.1–0.3 ng/ml. Single cell RNAseq libraries were prepared using the Nextera XT DNA Sample Preparation Kit (Illumina) and the Nextera Index Kit (Illumina) following the instructions in the Fluidigm manual ''Using the C1TM Single-Cell Auto Prep System to Generate mRNA from Single Cells and Libraries for Sequencing.'' Libraries were pooled, and 125bp paired-end sequencing was performed over two lanes of an Illumina HiSeq2000 instrument.
GEO Accession
GSM2844832