Pluto Bioinformatics

GSE140380: Transcriptional differences between skin from patients with atopic dermatitis and control skin obtained from the healthy margins of Mohs surgery patients

Bulk RNA sequencing

Purpose: To determine the transcriptional differences between skin from patients with atopic dermatitis and control skin obtained from the healthy margins of Mohs surgery patients; Methods: 4 mm skin biopsies of skin from atopic dermatitis patients or deidentified healthy margin surgical tissue were obtained and stored in RNA Later (-80C). Whole tissue RNA was isolated from tissue processed with a bead homogenizer using the QIAGEN RNeasy kit. Following DNase treatment (TurboDNase, Invitrogen), ribosomal RNA was removed (Ribo-zero kit, Illumina). mRNA was then fragmented in buffer containing 40 mM Tris acetate (pH 8.2), 100 mM potassium acetate, and 30 mM magnesium acetate and heated to 94C for 150 s. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Invitrogen, per manufacturers instructions) and random hexamers. A second strand reaction was performed to yield dscDNA. cDNA was blunt ended, had an A base added to the 30 ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina HiSeq-3000 using single reads extending 50 bases.; Samples were sequenced to an average depth of 32 million 1x50 reads on a HiSeq3000 (Illumina). Reads were aligned to Homo_sapiens reference build Ensembl_R76 using STAR, gene counts were determined with Subread:featureCount, transcript counts were produced by Sailfish, and sequence performance was assessed with RSeQC v2.3. SOURCE: Brian Kim (briankim@wustl.edu) - Washington University in Stl