title: 20666_m56_cell132
organism: Mus musculus
description: 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).
attributes.tag: ENA first public
attributes.value: 2018-08-06
attributes.tag: ENA last update
attributes.value: 2017-09-27
attributes.tag: ERCC measurement (percent)
attributes.value: 94.702278263
attributes.tag: External Id
attributes.value: SAMEA104324031
attributes.tag: INSDC center alias
attributes.value: Wellcome Trust Sanger Institute
attributes.tag: INSDC center name
attributes.value: Wellcome Trust Sanger Institute
attributes.tag: INSDC first public
attributes.value: 2018-08-06T17:03:14Z
attributes.tag: INSDC last update
attributes.value: 2017-09-27T18:13:49Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6072:20666_m56_cell132
attributes.tag: age
attributes.value: 6 to 14
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: CD4-positive, CD25-positive, alpha-beta regulatory T cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: Foxp3-IRES-GFP
attributes.tag: individual
attributes.value: C6
attributes.tag: library
attributes.value: m56
attributes.tag: library date
attributes.value: 03/06/2016
attributes.tag: organism part
attributes.value: skin
attributes.tag: phenotype
attributes.value: TCRb-positive, Foxp3-positive
attributes.tag: plate
attributes.value: 648
attributes.tag: position in library
attributes.value: F12
attributes.tag: position in smart-seq2
attributes.value: C6
attributes.tag: post analysis well quality
attributes.value: false
attributes.tag: sample name
attributes.value: E-MTAB-6072:20666_m56_cell132
attributes.tag: sex
attributes.value: male
attributes.tag: smart-seq2 date
attributes.value: 02/06/2016
attributes.tag: strain
attributes.value: C57BL/6
attributes.tag: total reads
attributes.value: 151552
attributes.tag: unmapped reads (percent)
attributes.value: 23.9231418919