title: Illumina HiSeq 2500 paired end sequencing; CCR8 expression defines tissue-resident memory T cells in human skin
design: CCR8 expression defines tissue-resident memory T cells in human skin
library_construction_protocol: All research involving human blood and tissue samples was approved by the local Research Ethics Committee. Written informed consent was obtained from each participant in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells were isolated from healthy donors via density gradient centrifugation using Lymphoprep (Axis- Shield). Human split skin samples (0.4 mm) were excised from healthy donors undergoing elective surgery using a dermatome. Sections were cut into 1 cm2 fragments and digested in a mixture of dispase II (1.25 U/ml), collagenase D (1 mg/ml), and DNase I (20 U/ml) (all from Roche) for 15–30 min at 37°C. The epidermis was separated from the dermis using forceps, and both the epidermis and dermis were subsequently cultured for 48 hr at 37°C in AB-RPMI. Single-cell suspensions of migrated cells were obtained from the culture medium by passing through a 40-μm pore mesh, washed, and resuspended in AB-RPMI. Total RNA was isolated using a Qiagen RNeasy Micro Kit (Qiagen), and cDNA was generated using a SMARTer Kit (ClonTech).  Libraries were prepared using a Nextera Kit (Illumina). Next-generation sequencing was performed using the Illumina HiSeq 2500 System with TruSeq Technology (Illumina).
attributes.tag: Experimental Factor: cell type
attributes.value: CD4-positive, alpha-beta T cell
attributes.tag: Experimental Factor: phenotype
attributes.value: CCR8 positive