Illumina HiSeq 2500 paired end sequencing; Single-cell transcriptomics analysis of Lipid A stimulated RAW264.7 cell line
Single-cell transcriptomics analysis of Lipid A stimulated RAW264.7 cell line
Single- cell sample collection and preparation was performed using the C1 Fluidigm platform, using the manufacturer's instruction. A suspension of appropriately stimulated 1x10^6 RAW 264.7 cells per ml was prepared in serum-free media and appropriately mixed with C1 suspension reagent. The resulting cell mixture was then loaded into C1 Single Cell AutoPrep IFC microfluidic chip (calibrated for medium 10-17μm cell sizes). The microfluidic chip was then placed into the C1 Fluidigm system for processing, using the 'mRNA Seq: Cell Load' script. Verification of single- cell capture was performed by wide-field microscopy. None RAW 264.7 murine macrophages  (obtained from ATCC) were cultured in Dulbecco's modified eagle medium supplemented with 10% foetal bovine serum (Gibco) and 1% non-essential amino acids as described previously (Bagnall et al., 2015). Cells were treated with 500ng/ml ipid A Salmonella Minnesota Re595 (VWR) for 3 hours (which included loading time for C1 prep cell capture).  Single- cell library construction was performed using the SMARTer Ultra Low RNA SMART-seq2 reagent kit (Takara®) for cDNA amplification, followed by the Nextera® XT DNA Index kKit for fragmentation and barcoding of samples (Illumina®).  Single- cell sample collection and preparation was performed using the C1 Fluidigm platform, using the manufacturer's instruction. Nextera® XT DNA Index Kit for fragmentation and barcoding of samples (Illumina®).
Experimental Factor: single cell identifier
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