T47D_d5_n1
Protocols: For 2D culture, cells were seeded in TC-treated culture flasks (Corning) and grown upon reaching 70 to 90% confluency. Cells were washed twice with pre-warmed Dulbecco´s phosphate-buffered saline (DPBS, Capricorn).  For 3D cultured cells, Coll I matrices were transferred to an empty well, washed twice with DPBS.  The human cell lines HCT-116 (colon carcinoma, ECACC 91091005), A549 (non-small cell lung cancer, ECACC 86012804), and T-47D (breast carcinoma, DSMZ ACC 739) were cultivated as recommended in McCoy's 5A, F-12K, and RPMI, respectively. Each medium was supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin. Cell cultivation and all incubation steps were performed at 37°C, 5% CO2 and 95% humidity. Unless otherwise stated, cell culture medium and consumables were used from Life Technologies. Cell lines were regularly tested negative for mycoplasma contamination by PCR (Takara Bio; Transgen Biotech) and authentication was confirmed by STR analysis (Eurofins).   For the 3D cultivation of single cells embedded in Coll I matrices, cells were carefully resuspended in 250 mM phosphate buffer (pH 7.5) and mixed with Coll I at 4°C to 2.5 mg/mL final concentration. The total volume of 300 µL was transferred to an ibitreat 24well plate (ibidi) and fibrillogenesis of Coll I was enabled at 37°C, 5% CO2 for 50 min to homogenously embed cells. Subsequently, Coll I matrices were washed twice with pre-warmed DPBS and 1 mL cell culture medium was added. Cells were cultivated for the indicated time (3, 5, 7, or 10 days). Cells were lysed and matrices were dissolved using 1 mL QIAzol Reagent (Qiagen). 200 µL chloroform (Roth) per mL QIAzol were added, followed by homogenization of the suspension and centrifugation at 4°C, 12000 x g for 15 min. The aqueous phase was merged with 1 volume cold isopropanol (Roth) and RNA was precipitated at - 20°C for 3 days. Precipitated RNA was pelleted at 4°C, washed three times with ice cold 75% ethanol (ITW Reagents). Afterwards, the pellet was dried and dissolved in RNase-free H2O. For library preparation, 100 ng of total RNA was used per sample whenever available. Samples T47D_d3_n1 (59 ng), A549_d3_n2 (89 ng), T47D_d3_n3Nr26 (81 ng), and A549_d3_n3 (67 ng) were processed using the maximum available input. Libraries were prepared using the KAPA RNA HyperPrep with RiboErase (HMR) kit together with KAPA Globin Depletion Hybridization Oligos (Roche) according to the manufacturer's instructions.
replicate
1
disease
invasive breast ductal carcinoma
organism
Homo sapiens
organism
Homo sapiens
collection date
not collected
scientific_name
Homo sapiens
cell type
breast invasive ductal carcinoma cell
common name
human
organism part
breast
isolate
not applicable
growth condition
3D
cell line
T47D
geographic location (country and/or sea)
not collected