PMEC4
Sus scrofa
Protocols: 4.4 × 105 PMECs were seeded and cultured in collagen-coated 6-well plates (1:10 collagen R in distilled water, Menal, Emmendingen, Germany) in complete medium without APS. Medium was changed after 24 h. At 90% confluency approximately 48 h after seeding, PMECs were challenged with 107/ml heat-inactivated E. coli for 3 h and for 24 h. The E. coli strain (gMEc240, sequence type 101, phylogroup B1, C+) was isolated from milk of PDS-positive sows and provided by the Institute of Microbiology and Epizootics, Department of Veterinary at the Freie Universität Berlin, Berlin, Germany. The unchallenged cells were used as a negative control. At each of the incubation time point, medium was discarded, and cells were washed three times with phosphate buffered saline (PBS, PAA) to remove the bacteria and then collected for DNA and total RNA isolation. 4.4 × 105 PMECs were seeded and cultured in collagen-coated 6-well plates (1:10 collagen R in distilled water, Menal, Emmendingen, Germany) in complete medium without APS. Medium was changed after 24 h. At 90% confluency approximately 48 h after seeding, PMECs were challenged with 107/ml heat-inactivated Escherichia coli (strain gMEc240, sequence type 101, phylogroup B1, C+) for 3 h and for 24 h. The DNA library was subjected to a bisulfite conversion using an EZ DNA Methylation Kit (Zymo Research). Genomic DNA was extracted from samples using a DNeasy kit (Qiagen) according to the manufacturer's recommendation including proteinase K digestion and RNase A treatment to obtain high purity DNA. 2 µg of DNA with a 1% spike-in control (unmethylated cl857 Sam7 Lambda DNA, Promega) was digested for 7 hours with 100U MspI (New England Biolabs) and then overnight with 50U TaqαI. The double-enzyme digested DNA fragments were end-repaired, A-tailed and ligated to TruSeq DNA adapters using a TruSeq Nano DNA library prep kit (Illumina). The adapter-ligated DNA fragments were size-selected to obtain inserts between 40 - 240bp. The DNA library was subjected to a bisulfite conversion using an EZ DNA Methylation Kit (Zymo Research). Library amplification was performed using a PfuTurbo Cx Hotstart DNA Polymerase kit (Stratagene). The quality of libraries was assessed on 2100 Bioanalyzer using a DNA chip (Agilent Technologies).
ENA-FIRST-PUBLIC
2019-07-17T17:03:33Z
ENA-LAST-UPDATE
2018-11-05T17:16:49Z
External Id
SAMEA5065626
INSDC center name
Leibniz-Institute for Farm Animal Biology (FBN) Institute of Genome Biology/ Genomics Unit
INSDC first public
2019-07-17T17:03:33Z
INSDC last update
2018-11-05T17:16:49Z
INSDC status
public
Submitter Id
E-MTAB-7363:PMEC4
broker name
ArrayExpress
cell type
mammary epithelial cell
common name
pig
organism part
mammary gland
sample name
E-MTAB-7363:PMEC4
scientific_name
Sus scrofa
sex
female