Tape strip collection, biopsy collection, RNA extraction, and RNA-seq
Twenty consecutive large D-Squame tape-strips (CuDerm, Dallas, Texas) were taken from the lesional skin of AD patients and from the skin of healthy controls. The first tape-strip was discarded, and remaining samples were frozen at –80oC as previously described.17,38 Four millimeters skin biopsies were taken from the lesional skin of AD patients and from the skin of controls (same subjects as tape-strips). RNA was extracted using an miRNAeasy Mini Kit (Qiagen, Hilden, Germany) as previously described.39 In tape-strips, RNA AmpliSeq libraries were constructed with the Ion AmpliSeq Transcriptome Human Gene Expression Kit by using 5 ng of RNA per sample. RNA-seq libraries were pooled and sequenced on the Ion S5 XL system sequencer with Ion 550 Chips (Thermo Fisher, Waltham, Mass). In biopsies, libraries were generated using TruSeq Stranded mRNA Library Prep kit (Illumina). Next generation sequencing was performed on Illumina NovaSeq6000 (Illumina Inc., 100 cycles, single-read sequencing).