MEC rep1
Mus musculus
Protocols: Mammary glands were harvested from a left and right pair of thoracic mammary gland number four using a slightly modified method from Cell Stem Cell, 2017, V20, issue 2, page 247-260.Tissue was minced and then dissociated overnight in gentle dissociation solution. Single cell suspensions were generated. Mammary epithelial cells were enriched using EasySep Mouse Epithelial Cell Enrichment Kit from Stem Cell Technologies.MECs were seeded into tissue culture flasks in Epicult-B medium with supplements (Stem Cell Technologies) and 10% FBS. 3 days later, the medium was replaced with serum-free medium to reduce the growth of contaminating stromal cells. Cells were harvested by trypsinisation. Total RNA from cells were extracted using RNeasy Mini kit 74104 Qiagen, Manchester, UK), cDNA library preparation was carried out at Oxford Genomics Centre, The Wellcome Trust Centre for Human Genetics using PolyA+ RNA enrichment method for total RNA from cultured cells . mRNA fraction was selected from the total RNA before conversion to cDNA. Second strand cDNA synthesis incorporated dUTP. The cDNA was end-repaired, A-tailed and adapter-ligated. Prior to amplification, samples underwent uridine digestion. The prepared libraries were size selected, multiplexed and quality checked.