title: Illumina HiSeq 4000 sequencing; Single cell RNA-seq of human breast skin cells
design: Single cell RNA-seq of human breast skin cells
library_name: 4820STDY7389012_p
library_construction_protocol: 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.
attributes.tag: Experimental Factor: inferred cell type
attributes.value: keratinocyte CD49f-