GSM9037416: Animal 502, Late Pregnancy; Bos taurus; RNA-Seq
Tissue was minced to a fine slurry in freshly prepared Collagenase solution (0.2% Collagenase A (Roche), 0.05% Trypsin (1:250 powder, 100U/ ml Gibco/life sciences), Hyaluronidase (Sigma, St Louis MO), 5% Fetal Calf serum, Pen/Strep/Fung (hyclone) or 5µg/ml Gentamycin (Sigma), in DMEM/F12 (Gibco/Life technologies) 10 ng/ml Insulin). Minced tissue was incubated in Collagenase solution (10 ml solution/g tissue) for 3.5 hours at 37C in 50 ml conical tube with slow shaking (120 rpm). Digested tissue was centrifuged for 10 minutes at 453xg at 4°C, supernatant and fat layer discarded, pellet was gently resuspended by pipetting in 5 ml DMEM/F12 without serum. A further 5 ml DMEM/F12 without serum was added and resuspended sample was centrifuged for 10 minutes at 453xg at 4°C, media was discarded and pellet resuspended gently in 10 ml DMEM/F12 and centrifuged for 10 minutes at 453xg at 4°C . Pellet was resuspend gently in 10 ml followed by brief pulls spins at 453xg (centrifuge set to 1 minute @ 453 x g, hit the brakes 2 seconds after 453 xg is reached). Pulls spins were repeated at least 4 time or until very few single cells and mainly epithelial organoid clusters were observed under a microscope. Organoid pellet for RNA-analysis was resuspended in 1 ml TRIzol and stored at -80C. High-quality total RNA (RIN > 7) was extracted from frozen mammary epithelial organoid pellets in TRIzol using the NucleoSpin® miRNA isolation kit (MACHEREY-NAGEL) according to the manufacturer's protocol. Large and small RNA fractions (< 200nt) were isolated separately. The large RNA fraction was used for mRNA-seq library preparation. mRNA-seq libraries were prepared from the large RNA fraction. Sequencing was performed as follows: Virgin & Peak Lactation: 100 bp paired-end, stranded. Mid-pregnancy, Late-pregnancy, Early Lactation: 5/7 samples: 125 bp paired-end, non-stranded; 2/7 samples: 75 bp single-end, stranded.