eG2.2
Mus musculus
Protocols: The following mouse C57BL/6 (B6) primary cell lines were purchased and cultured for RNA isolation according to manufacturer's instructions. Bone Marrow Mononuclear cells, c57-6271F, Caltag Medsystems, Buckingham, UK; Mouse Embryonic Fibroblasts, untreated, GSC-6002, AMS Biotechnology Ltd; Mesenchymal Stem cells, MUBMX-01001, Cambridge Bioscience, Cambridge, UK; embryonic stem cell line #693 ES C57BL/6 (SCRC-1002, from LGC standards, Middlesex, UK). Embryonic mammary progenitor cell (eMPC) clones were derived from E12.5-stage mammary organs. After microdissection, mammary primordial tissue was cultured on thick basement membrane extract for 4 weeks. After enzymatic dissociation eMPCs were plated and expanded in 2D culture to obtain a pool of eMPCs. eMPC cells were subject to single cell sorting using FACS. Clones were expanded as single-cell derived clones (eMPC1, 2, etc.) to create the eighteen single cell-derived clones described in this study. 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.