20666_m34_cell20
Mus musculus
Protocols: Mouse melanoma induction: The melanoma induction experiments were performed in accordance with UK Home Office regulations under Project License PPL 80/2574. Protocol used was adapted from a previous publication (Riedel et al., 2016). For syngeneic tumours, 2.5 × 105 B16.F10 melanoma cells were inoculated subcutaneously into the shoulder region 6- to 14-week-old female Foxp3-IRES-GFP mice (Haribhai et al., 2007). Animals were excluded only if tumours failed to form or if health concerns were reported. Control Foxp3-IRES-GFP mice were injected with 50 μl PBS. Animals were culled after 11 days. Tumour tissue, tumour-draining (brachial) lymph nodes and spleen were isolated for subsequent analysis. PBS-injected and steady-state skin, draining lymph nodes (bLN) and spleen were collected from control mice. Tumour and PBS-injected skin were mechanically disrupted and digested in a 1ml mixture of 1 mg/ml collagenase A (Roche) and 0.4 mg/ml DNase I (Roche) in PBS (solution A) at 37°C for 1h with 600rpm rotation. 1ml of PBS containing 1mg/ml Collagenase D (Roche) and 0.4 mg/ml DNase I (solution B) was then added to each sample, which returned to 37 °C for 1h with 600 rpm rotation. Lymph nodes were digested for 30min in 500μl of solution A, and for further 30min after the addition of 500μl of solution B. EDTA at the final concentration of 10mM was added to all samples. Spleens were processed as described previously (Uhlig et al., 2006). Suspensions were passed through a 70 µm mesh before immunostaining with combinations of fluorescently conjugated antibodies. Samples from different animals were kept separated throughout processing and sorting. Cells from PBS-injected and steady-state skin were compared and concluded to be equivalent in expression. Mouse and human cell suspensions were stained with the antibodies in Supplementary Table 1 and DAPI. 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. Smart-seq2 protocol was largely followed to obtain mRNA libraries from single-cells. Oligo-dT primer, dNTPs (ThermoFisher, cat. no. 10319879) and 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 published21, using 50U of SMARTScribe™ Reverse Transcriptase (Clontech, cat. no. 639538). The cDNA libraries for sequencing were prepared using Nextera XT DNA Sample Preparation Kit (Illumina, cat. no. FC-131-1096), according to the protocol supplied by Fluidigm (PN 100-5950 B1).
ENA first public
2018-08-06
ENA last update
2017-09-27
ERCC measurement (percent)
3.2364195447
External Id
SAMEA104324346
INSDC center alias
Wellcome Trust Sanger Institute
INSDC center name
Wellcome Trust Sanger Institute
INSDC first public
2018-08-06T17:03:18Z
INSDC last update
2017-09-27T18:13:59Z
INSDC status
public
Submitter Id
E-MTAB-6072:20666_m34_cell20
age
6 to 14
broker name
ArrayExpress
cell type
CD4-positive, CD25-positive, alpha-beta regulatory T cell
common name
house mouse
disease
melanoma
genotype
Foxp3-IRES-GFP
individual
T5
library
m34
library date
03/06/2016
organism part
skin
phenotype
TCRb-positive, Foxp3-positive
plate
641
position in library
A20
position in smart-seq2
A10
post analysis well quality
true
sample name
E-MTAB-6072:20666_m34_cell20
sex
male
smart-seq2 date
01/06/2016
strain
C57BL/6
total reads
1758036
unmapped reads (percent)
4.7435888685