ctrl_rep1
Homo sapiens
Protocols: The 6-well tissue culture plate was put on ice and media was removed from each well. Next, each well was washed with 1 ml of ice-cold PBS. Cells were then harvested in 1 ml of ice-cold PBS using a cell scraper and next collected by centrifugation at 500xg for 5 min at 4°C. The supernatant was subsequently removed and the cell pellet was either snap-frozen and stored or immeadiately subjected to nucleic acid extraction. MCF7 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% foetal bovine serum at 37°C with 5% CO2. One well of a 6-well tissue culture with 90% confluent cells was transfected with 2 µg of a minigene library one day prior to harvesting using 20 µg polyethylenimine MW ~ 2500 transfection reagent (Polysciences, Inc.). The RON minigene was cloned into the pcDNA 3.1 (+) vector (Invitrogen). The minigene construct contains a sequence of the MST1R gene including exon 11 together with the complete flanking introns and the constitutive exons 10 and 12 (chr3: 49,933,098 - 49,933,837, GRCh37/hg19). The RON minigene library comprises of approximately 6,000 minigenes with three to four mutations on average per minigene. RNA extraction was performed using the QIAGEN RNeasy plus kit according to the manufacturer's instructions. 17 µg of total RNA was used for poly(A)-selection using magnetic Dynabeads® Oligo (dT)25 from Thermo Fisher Scientific according to the manufacturer's instructions. 400 - 500 ng of the resulting poly(A) selected RNA was used for cDNA synthesis using the RevertAid reverse transcriptase from Thermo Fisher Scientific according to the manufacturer's instructions. The resulting cDNA was used as a template for PCR amplification via an emulsion PCR protocol as described by Williams et al., 2006. The following steps were then performed to clean the water-in-oil emulsion: First, the water phase was extracted by washing twice with 1 ml of water-saturated diethyl ether. Second, residual solvent from the broken emulsion was removed by centrifugation under vacuum for 12 min at 30°C.Finally, PCR products were purified using 2 volumes of Agencourt® AMPure® XP beads from Beckman Coulter according to the maufacturer's instructions.
ENA first public
2018-06-27
ENA last update
2017-11-21
External Id
SAMEA104394258
INSDC center alias
Institute of Molecular Biology (IMB)
INSDC center name
Institute of Molecular Biology (IMB)
INSDC first public
2018-06-27T17:02:54Z
INSDC last update
2017-11-21T13:48:49Z
INSDC status
public
Submitter Id
E-MTAB-6217:ctrl_rep1
broker name
ArrayExpress
cell line
MCF-7
cell type
epithelial cell
common name
human
disease
invasive ductal carcinoma
genotype
pool of minigene library MST1R construct
metastatic site
pleural effusion
organism part
mammary gland
sample name
E-MTAB-6217:ctrl_rep1