GSM3103253: WT_E_3: RSK2(+/+) WT Estrous; Mus musculus; RNA-Seq
4th mammary glands were isolated from donor mice, minced, and digested in DMEM/F12 supplemented with 2mg/ml Collagenase A and 100U/ml Pen/Strep for 2.5h in 37oC 5% CO2 incubator.  Digested material was pelleted at 180g for 5 min and the pellet was resusupended in DNAse I (1000U/ml) for 3-5 min in 37oC 5% CO2 incubator.  Fetal bovine serum (FBS) was added and the digested tissue was pelleted at 180g for 10 min.  The pellet was washed with phosphate-buffer saline, pelleted, resuspended in Accumax and placed in Thermomixer at 37oC for 10 min.  Digested material was pelleted at 180g for 3 min, resuspended in 5x trypsin for 5 min at 37oC.  Trypsin was quenched with FBS and cells were pelleted and resuspended in PBS or DMEM/F12.  The cell preparation was filterd through 70-mm mesh to obtain single cell suspension. Cells were stained with primary Ab: EpCam, CD49f, Sca1, and CD49b and sorted using FACSAriaII. For RNA isolation, 5x10^4 EpCAMhiCD49f+Sca1+Cd49b- cells were FACS sorted and total RNA extraction (RNeasy Micro Kit) was performed. The RNA quality was tested using Agilent 100 Bioanalyzer (RIN 8) Library constructions were performed using TruSeq RNA Library Prep Kit (Illumina)