Illumina HiSeq 2500 paired end sequencing; Single-cell RNA sequencing reveals a dynamic stromal niche within the evolving tumour microenvironment
Single-cell RNA sequencing reveals a dynamic stromal niche within the evolving tumour microenvironment
Animals were housed in accordance with UK regulations and experiments were performed under project licences PPL 80/2574 or PPL P8837835. The C57BL/6 derived B16.F10 melanoma cell line was purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle medium (DMEM, Life Technologies), supplemented with 1% Penstrep and 10% FBS. 2.5 x105 B16 cells were injected, subcutaneously, into the shoulders of either wild type (WT) C57BL/6 mice, or C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ mice (Jackson Laboratory). After 5, 8 and 11 days animals were sacrificed and tissues collected for analysis. In addition, skin was also taken from non-tumour bearing mice. Tumours were mechanically dissociated and digested in 1mg/ml collagenase D (Roche), 1mg/ml collagenase A (Roche) and 0.4mg/ml DNase (Roche) in PBS, at 37OC for 2hs. Lymph nodes were mechanically dissociated and digested with 1mg/ml collagenase A (Roche) and 0.4mg/ml DNase (Roche) in PBS, at 37OC. After 30 mins, Collagenase D (Roche) was added (final concentration of 1mg/ml) to lymph node samples and digestion was continued for a further 30 mins. EDTA was added to all samples to neutralise collagenase activity (final concentration (5mM) and digested tissues were passed through 70μm filters (Flacon) ready for staining. Single cells were isolated from processed tissues using fluorescence-activated cell sorting (FACS). Once processed, samples were incubated with a fixable fluorescent viability stain (Life Technologies) for 20mins (diluted 1:1000 in PBS) prior to incubation with conjugated primary antibodies for 30 mins at 4OC. Antibodies were diluted in PBS 0.5% BSA according to table SX. Stained samples were index sorted, using the BD influx flow cytometer system, Single-cells were sorted in 2μl of Lysis Buffer (1:20 solution of RNase Inhibitor (Clontech, cat. no. 2313A) in 0.2% v/v Triton X-100 (Sigma-Aldrich, cat. no. T9284)) in 96 well plates, spun down and immediately frozen at -80 degrees. Reverse transcription and cDNA pre-amplification were performed according to the SmartSeq2 protocol 49 to obtain mRNA libraries from single-cells. Oligo-dT primer, dNTPs (ThermoFisher, cat. no. 10319879) and an ERCC RNA Spike-In Mix (1:50,000,000 final dilution, Ambion, cat. no. 4456740) were then added. Reverse Transcription and PCR were performed as previously published 49, using 50U of SMARTScribe™ Reverse Transcriptase (Clontech, cat. no. 639538). cDNA libraries were prepared using the Nextera XT DNA Sample Preparation Kit (Illumina, cat. no. FC-131-1096), according to the protocol supplied by Fluidigm (PN 100-5950 B1).
Experimental Factor: single cell identifier
21784_6_156
Experimental Factor: genotype
wild type genotype
Experimental Factor: phenotype
CD45+ CD3e- B220- CD11b+