Illumina NovaSeq 6000 paired end sequencing; The impact of the HSPA2 gene knockout on transcriptome profile in immortalized epidermal keratinocytes (HaCaT line)
The impact of the HSPA2 gene knockout on transcriptome profile in immortalized epidermal keratinocytes (HaCaT line)
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. CRISPR/Cas9-modified HaCaT cells were plated onto polycarbonate membranes of the transwell cell culture inserts coated with collagen-IV/fibroblast matrix 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 CRISPR-Cas9 gene editing system to generate HSPA2-null isogenic clones. In brief, cells were transfected with HSPA2 Double Nickase Plasmid (Santa Cruz Biotechnology) and Lipofectamine 3000. Transfected cells were selected in a standard culture medium (high glucose DMEM/FBS) containing puromycin (1 µg/mL). Afterwards, single clones were obtained by limiting dilution on 96-well plates. Disruption of the HSPA2 gene in clones was examined at protein level by Western blot and confirmed by Sanger sequencing. Six individual isogenic clones with the non-edited HSPA2 gene sequence were pooled as control CRISPR-CTR clones; four isogenic gene-edited clones with mutated HSPA2 sequence and a full protein loss were pooled and named CRISPR-4, pool containing two different HSPA2-null clones was named CRISPR-2B. 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.