Homo sapiens
Protocols: 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.
ENA-FIRST-PUBLIC
2023-07-24T00:22:16Z
ENA-LAST-UPDATE
2023-07-24T00:22:16Z
External Id
SAMEA113987030
INSDC center alias
Maria Sklodowska-Curie National Research Institute of Oncology Gliwice Branch
INSDC center name
Maria Sklodowska-Curie National Research Institute of Oncology Gliwice Branch
INSDC first public
2023-07-24T00:22:16Z
INSDC last update
2023-07-24T00:22:16Z
INSDC status
public
Submitter Id
E-MTAB-13166:S4101Nr9
broker name
ArrayExpress
cell_line
HaCaT
collection date
not collected
common name
human
disease
normal
genotype
CRISPR/Cas9 mediated HSPA2 knockout gDNA 2B
geographic location (country and/or sea)
not collected
growth condition
ALI culture
isolate
not applicable
organism part
skin
phenotype
CRISPR/Cas9 mediated HSPA2 knockout
replicate
CRISPR-2B
sample name
E-MTAB-13166:S4101Nr9
scientific_name
Homo sapiens