Illumina HiSeq 2500 paired end sequencing; Single CD4+ cells after Nippostrongylus brasiliensis infection
Single CD4+ cells after Nippostrongylus brasiliensis infection
Three small (5-10 μm) C1 Single-Cell Auto Prep IFC chips (Fluidigm) were primed and 5,000 cells were sorted directly into the chip. To allow estimation of technical variability, 1 μl of 1:4000 dilution ERCC (External RNA Controls Consortium) spike-in mix (Ambion, Life Technologies) was added to the lysis reagent. Cell capture sites were visually inspected one by one using a microscope. C57BL/6 female mice were subcutaneously injected with 100ul (300/500 live third stage Nippostrongylus Brasiliensis larvae per dose), over two sites. Mediastinal lymph nodes, mesenteric lymph nodes, and lungs were taken from infected mice, 5 days after infection. Cells were isolated from mediastinal lymph nodes and mesenteric lymph nodes, by smashing the tissue though 70μm cell strainer. Lungs were incubated in collagenase D (0.72mg/ml, Amersham, Bucks, UK) for 30min, smashed though 70μm cell strainer, and suspended in RBC lysis buffer (eBioscience Ltd). Single cells were then stained with CD3e-APC-cy7, CD4-PE-Cy7 and CD62L-Violet-BV510 for 30 minutes on ice and then were washed.  Reverse transcription and cDNA preamplification were performed using the SMARTer Ultra Low RNA kit (Clontech). Sequencing libraries were prepared using Nextera XT DNA Sample Preparation kit with 96 indices (Illumina), according to the protocol supplied by Fluidigm.
Experimental Factor: lung
organism part
Experimental Factor: 27
single cell ID