GSM5589715: Luminal_4; Mus musculus; RNA-Seq
Mammary gland was dissected from 6-8 weeks old mice (used 4 mice for each experiment), and cut in pieces of 1 mm^3. To isolate mammary glands, tissue was incubated in enzyme digestion buffer (Advanced DMEM/F12 (Thermo Fisher Scientific), 2 mg/ml Collagenase Type VIII (Merck), 5 μg/ml Insulin (Merck), 50 μg/ml Gentamicin (Thermo Fisher Scientific), 20 mg/ml Trypsin (Thermo Fisher Scientific), 5 % Fatal bovine serum (FBS) (Biowest)) at 37 oC for 30 min, and centrifuged at 500 G for 10 minutes. After removing fat fraction, the pellet was re-suspended in TrypLE Express (Thermo Fisher Scientific) with 10 μM Y-27632 (Miltenyi Biotec) and incubated at 37 oC for 10 minutes. DMEM/F12 with 10 % FBS was added in cell suspension, and cell suspension was filtered through 30 μm cell-strainer (Miltenyi Biotec). After centrifugation, the pellet was resuspended in HBSS (Merck) with 2% FBS, and incubated with PE-Cy7-conjugated anti-CD24 (clone M1/69, BD Pharmingen), APC-conjugated anti-CD29 (clone eBioHMb1-1, Thermo Fisher Scientific), PE-conjugated anti-CD45 (clone 30-F11, Thermo Fisher Scientific), PE-conjugated anti-CD31 (clone MEC 13.3, BD Pharmingen), PE-conjugated anti-CD140a (clone APA5, Thermo Fisher Scientific) and APC-Cy7- conjugated anti-CD326 (Ep-CAM) (clone G8.8, BioLegend) at 4 oC for 30 minutes. After wash in HBSS with 2% FBS, the cells were resuspended in HBSS with 2% FBS and 1 μg/ml DAPI (Merck). Cells were sorted on FACS Aria with FACS DiVa software (BD Bioscience), and were collected in HBSS with 2% FBS. RNA was isolated from sorted epithelial cells, organoids or MCF7 cells using NucleoSpin RNA XS (Macherey-Nagel) or RNeasy mini kit according to the manufacturer's instructions. RNA were sent to Beijing Genomics Institute (BGI) for transcriptome library preparation and sequencing.