S2952Nr2_p
Each culture insert was washed twice with ice-cold calcium/magnesium free PBS and next the epidermis was submerged with RNA extraction buffer and scraped off the polycarbonate membrane using rubber tipped cell scraper. The suspension was immediately snap-frozen in dry ice and stored at -70°C until the total RNA extraction procedure. Lentivirus-mediated shRNA modified HaCaT cells were plated onto polycarbonate membranes of the transwell cell culture inserts coated with extracellular matrix protein and maintained in a standard DMEM high glucose medium supplemented with 10% FBS in submerged culture for 72 h. Subsequently, the cells were exposed to the air–liquid interface (ALI) culture by removing the medium in the upper compartment; while the standard DMEM high glucose/FBS medium supplemented with 2 ng/ml TGFα was placed under the filter. The medium was renewed every two days during the 18 days of ALI culture. Wild type HaCaT cells were modified by lentivirus-mediated shRNA method to generate HSPA2-depleted cell lines. The shRNA sequence (shRNA-4) targeted the coding sequence of human HSPA2 (Gene Entrez ID: 3306), was selected from the Mission shRNA Library (Sigma–Aldrich). shRNA-luc sequence targeting the firefly luciferase (luc) gene was used as a control nontargeting shRNA. Sense and antisense oligonucleotides were annealed and inserted into the pLVX-shRNA1 (bearing a puromycin resistance gene)  vector (Clontech/Takara Bio) at the BamHI/EcoRI site. Infectious lentiviruses were generated by transfecting shRNA-encoding plasmids into HEK293T packaging cells according to the manufacturer's instructions (Clontech/Takara Bio, Lenti-X shRNA Expression System). Virus-containing supernatants were collected 48 h after transfection and a viral titer was confirmed using Lenti-X GoStix (Clontech/Takara Bio). Cells were transduced using lentiviruses-containing supernatants for 24 hr at 37°C with the addition of polybrene according to the manufacturer's recommendations. A comparable number of HaCaT cells remained viable after transduction with non-targeting or HSPA2-targeting lentiviral vectors and survived puromycin (1 µg/mL) selection for the establishment of stably transduced cell lines. Total RNA was extracted using the GeneJET RNA Purification Kit in accordance with the manufacturer's protocol. RNA samples were purified from contaminating DNA by treatment with RNase-free DNase I, possible DNA contamination was examined also by a control PCR reaction. 100 ng RNA was used for library preparation Sequencing library was prepared according to the standard Illumina protocol using the TruSeq Stranded mRNA library preparation kit.