GSM7505531: n76_nonresponder_dermis; Homo sapiens; RNA-Seq
Skin punch biopsy samples were rinsed in sterile phosphate buffered saline without calcium or magnesium (HyClone Laboratories, GE Healthcare) and then incubated at room temperature in a 3.8% ammonium thiocyanate (Sigma Aldrich) in phosphate buffered saline without calcium or magnesium solution for 45 minutes. The epidermis and dermis were subsequently separated using sterile forceps, placed into separate RNase-free tubes containing 700 μL QIAzol (Qiagen), homogenized by hand using RNase-free pestles (Argos), and placed at −80 °C. All samples were collected, processed under sterile conditions, and stored within a 2-hour time frame. RNA was isolated using chloroform (Fisher Scientific), RNeasy Mini Kit (Qiagen), and RNase-free DNaseI (Qiagen). Briefly, 140 μL chloroform was added to each tube, the mixture was vortexed for 5 seconds, and the tubes were left to stand for 2 to 3 minutes at room temperature. After centrifugation at 12 000 g for 15 minutes at 4 °C, the top aqueous layer was transferred to a new tube and combined with an equal volume of 100% ethanol. Thereafter, the Qiagen RNeasy Mini Kit protocol was followed, ending with a 35 μL elution volume of RNase-free water. RNA quality was assessed on a Synergy H1 microplate reader (BioTek Instruments) with UV readings of 260 nm, 280 nm, 230 nm, and 320 nm. Purified RNA was stored at −80 °C until downstream analysis. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers,  followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.  For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.