Comment[ArrayExpressAccession]	E-MTAB-8142
MAGE-TAB Version	1.1
Comment[Submitted Name]	Single cell RNA-seq of human breast skin cells				
Investigation Title	Single cell RNA-seq of human breast skin cells
Experiment Description	Cells were generated for a single cell atlas of adult human skin to dissect the cellular and molecular organisation underpinning human skin immune barrier function. Surplus skin from breast reconstruction surgery was collected (n=3), then the top 200 μm layer was taken. Epidermis was separated from the dermis, and both layers were separately digested. Single cells were FACS sorted into eight categories, then loaded onto a 10x Genomics Chromium Controller, and sequenced on an Illumina HiSeq 4000.
Experimental Factor Name	sampling site	FACS sorting	disease
Experimental Factor Type	sampling site	FACS sorting	disease
Experimental Factor Term Source REF
Experimental Factor Term Accession Number
Person Last Name	Polanski	Vegh	Haniffa
Person First Name	Krzysztof	Peter	Muzlifah
Person Mid Initials
Person Email	kp9@sanger.ac.uk	peter.vegh@newcastle.ac.uk	m.a.haniffa@newcastle.ac.uk
Person Phone
Person Fax
Person Address	Wellcome Genome Campus Hinxton, Cambridgeshire, CB10 1SA, UK	Institute of Cellular Medicine Newcastle University Medical Sch, Framlington pl Newcastle upon Tyne, NE2 4HH United Kingdom	Institute of Cellular Medicine Newcastle University Medical Sch, Framlington pl Newcastle upon Tyne, NE2 4HH United Kingdom
Person Affiliation	Sanger Institute	Newcastle University	Newcastle University
Person Roles	submitter	submitter	investigator
Date of Experiment	2018-02-01
Public Release Date	2021-02-10
PubMed ID
Publication DOI
Publication Author List
Publication Title
Publication Status
Publication Status Term Source REF
Publication Status Term Accession Number
Protocol Name	P-MTAB-87566	P-MTAB-87567	P-MTAB-87568	P-MTAB-87569	P-MTAB-87570
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO
Protocol Term Accession Number	EFO_0005518	EFO_0002944	EFO_0004184	EFO_0004170	EFO_0003816
Protocol Description	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.	The libraries from eight loaded channels, four dermal and four epidermal, from each skin sample were multiplexed together and sequenced on an Illumina HiSeq 4000. Each set of eight libraries were distributed over eight lanes of the flow cell and sequenced using the following parameters: Read1: 26 cycles, i7: 8 cycles, i5: 0 cycles; Read2: 98 cycles to generate 75bp paired end reads.	Droplet-based (10x) sequencing data was quantified using the Cell Ranger Single-Cell Software Suite (version 2.0.2, 10x Genomics Inc) and aligned to the GRCh38 reference genome (official Cell Ranger reference, version 1.2.0).
Protocol Hardware				Illumina HiSeq 4000
Protocol Software
Term Source Name	EFO
Term Source File	http://www.ebi.ac.uk/efo/
Term Source Version
Comment[AEExperimentType]	RNA-seq of coding RNA from single cells
Comment[TemplateType]	Human - Single-cell sequencing
Comment[AdditionalFile:txt]	arrayexpress_metadata.txt
Comment[EAExpectedClusters]
Comment[EAExperimentType]	differential
Comment[EAAdditionalAttributes]	individual	developmental stage
Comment[EACurator]	SF
Comment[SecondaryAccession]	ERP149679
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SDRF File	E-MTAB-8142.sdrf.txt