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).