SKN8090563
Homo sapiens
Protocols: Surplus skin from breast reconstruction surgery was collected in accordance with the Newcastle and North Tyneside 1 Research Ethics Committee at the Royal Victoria Infirmary, Newcastle upon Tyne NHS Foundation Trust (Newcastle Dermatology Biobank - REC reference: 08/H0906/95+5). Skin was cut to thin strips in PBS and the top 200 μm layer was taken using a dermatome with a Pilling Wecprep blade and a .008 gauge Goulian guard. A grid of slits was cut into the skin sheets to aid enzymatic access, then the skin was treated with 2U/ml dispase II (Roche, 04942078001) in RPMI at 37oC for 1 hour. The epidermis was peeled from the dermis, and both fragments were separately digested in a petri-dish at 37oC 5% CO2 in RF-10 media with 1.6 mg/ml type IV collagenase (Worthington, CLS-4) overnight (roughly 12 hours). All work was done within class II biological safety cabinets using autoclave-sterilised equipment. The media was then collected with a serological pipette and filtered through a sterile 100 μm cell strainer (BD Falcon, 352360). The petri dish and strainer were washed through with RF-10 to collect any remaining cells. Cells were pelleted by centrifuging at 500 x g for 5 minutes. Supernatant was discarded and the pellet resuspended in 1 ml RF-10 by gently pipetting up and down. Cells were then counted by taking off 10 μl, mixing 1:1 with 0.4% trypan blue (Sigma, T8154) to stain dead cells and counting on a haemocytometer. 7000 live, single cells were loaded on to each of the Chromium Controller (10x Genomics, Pleasanton, CA, USA), then cDNA synthesis and amplification were performed as per the 10x Genomics protocol. Sequencing libraries were generated using the Single Cell 3' Reagent Kit as per the 10x Genomics protocol.
ENA-FIRST-PUBLIC
2023-07-21T12:16:50Z
ENA-LAST-UPDATE
2023-07-21T12:16:50Z
External Id
SAMEA114135158
FACS sorting
live single
INSDC center alias
Sanger Institute
INSDC center name
Sanger Institute
INSDC first public
2023-07-21T12:16:50Z
INSDC last update
2023-07-21T12:16:50Z
INSDC status
public
Submitter Id
E-MTAB-8142_2:SKN8090563
broker name
ArrayExpress
collection date
not collected
common name
human
developmental stage
adult
disease
atopic eczema
geographic location (country and/or sea)
not collected
individual
E1
isolate
not applicable
organism part
skin of body
sample name
E-MTAB-8142_2:SKN8090563
sampling site
dermis
scientific_name
Homo sapiens
sex
male