Colon3
Protocols: Colons were removed and washed in PBS/BSA. Excess fat and contents were removed from colons then tissues were opened longitudinally and cut into sections. Colon tissue was washed twice for 25 min in RPMI/BSA/EDTA at 37oC in a shaking incubator. Colon tissue was digested with RPMI/FCS/collagenase VIII/DNase1 for 45 minutes at 37oC in a shaking incubator. Tissue digests were passed through a 70 μm filter and washed in PBS/BSA/EDTA. Tissue digests were separated by centrifugation on a 40%/80% Percoll gradient and cells collected at the interface. Single cell suspensions were washed in PBS/BSA/EDTA and stained with fluorochrome-conjugated antibodies in PBS/BSA/EDTA for 20 min at 4 oC in the dark. Cells were washed and resuspended in PBS/BSA/EDTA/DNase1 for FACS sorting. Foxp3+ Tregs were sorted as live CD45+Lineage-CD4+TCRβ+Foxp3-YFP+ cells. Lineage included CD8, TCRγδ, B220, CD11b, CD11c, Gr1. Dead cells were excluded using DAPI. For gene expression analysis by RNA sequencing, small bulks of 300 Treg cells were sorted into 2μL lysis buffer (0.8% Triton X-100 and 2 U/μL RNase inhibitor) in a 96-well plate on ice Sequencing libraries were prepared following the Smart-seq2 protocol (Picelli et al., 2013) with own in-house indexing primers (Lamble et al., 2013)
ENA first public
2021-08-01
ENA last update
2021-08-01
External Id
SAMEA7202709
INSDC center alias
OXFORD
INSDC center name
University of Oxford
INSDC first public
2021-08-01T00:17:23Z
INSDC last update
2021-08-01T00:17:23Z
INSDC status
public
Submitter Id
E-MTAB-9477:Colon3
age
11 to 14
broker name
ArrayExpress
cell type
regulatory T cell
common name
house mouse
genotype
wild type genotype
individual
300 cells from 5 pooled male and female mice
organism part
colon
sample name
E-MTAB-9477:Colon3
sex
mixed