Illumina HiSeq 2500 paired end sequencing; Simultaneously inferring T cell fate and clonality from single cell transcriptomes
Simultaneously inferring T cell fate and clonality from single cell transcriptomes
Female C57BL6/N mice aged 6-8 weeks were infected intravenously with 0.2 ml Salmonella Typhimurium M525 containing 5x105 CFU of bacteria in sterile phosphate buffered saline (PBS, Sigma-Aldrich). At day 49 post infection (p.i.) mice were sacrificed with spleens and livers being harvested. Bacteria were enumerated from the livers by serial dilution and plating onto agar plates (Oxoid) to confirm levels of infection. Single-cell suspensions were prepared by homogenising spleens through 70 m strainers and lysing erythrocytes. Following incubation with CD16/CD32 blocking antibody, the cells were stained with titrated amounts of fluorochrome conjugated antibodies for CD44(FITC), CD127(PE), CD62L(PE-CF594), TCR(PerCP-Cy5.5), CD161(APC), CD8(APC-H7), CD4 (BV510), CD8(APC-H7), CD161(BV421), and CD4(BV510). Cells were sorted as CD4+ TCRB+ CD161- CD44+ CD62LLow CD127Hi. Capture and processing of single CD4+ T cells was performed using the Fluidigm C1 autoprep system. Cells were loaded at a concentration of 1700 cells l-1 onto C1 capture chips for 5-10 m cells. ERCC (External RNA Controls Consortium) spike-in RNAs (Ambion, Life Technologies) were added to the lysis mix. 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: Salmonella typhimurium
infect
Experimental Factor: 49
time
Experimental Factor: cell 159
single cell ID