GSM6730045: Mammary fibroblasts co-cultured with macrophages repetition1; Mus musculus; RNA-Seq
Normal mammary fat pad and mesometrial fat fibroblasts were isolated and dissociated from the mammary fat pads or fat tissue of two BALB/c females per each biological replicate. Tissue was minced and dissociated using a gentle MACS dissociator, in the presence of an enzymatic digestion solution containing 1 mg/ml collagenase II (Merck Millipore, 234155), 1 mg/ml collagenase IV (Merck Millipore, C4-22) and 70 U/ml DNase (Invitrogen, 18047019) in Dulbecco's modified Eagle's medium (DMEM). The samples were filtered through a 70-μm cell strainer into cold PBS and cells were pelleted by centrifugation at 350g for 5 min at 4 °C and resuspended in red blood cell lysis buffer (BioLegend 420301), then washed with PBS and centrifuged at 350g for 5 min at 4 °C. Mammary and fat fibroblasts were seeded on collagen I (Cat. C3867, Sigma-Aldrich, Rehovot, Israel) coated 10cm or 6-well plates, respectively. The cells were expanded for 6 days in DMEM with 5% P/S and 10% of FBS and the media was replaced every 3 days.    Lungs of BALB/c female were excised, dissociated, minced, and incubated with enzymatic digestion solution containing 3 mg/ml collagenase A (Sigma Aldrich, 11088793001) and 70 unit/ml DNase in RPMI 1640 (Biological industries, 01-100-1A) using a gentleMACS dissociator, 30 min at 37oC. The samples were filtered through a 70-μm cell strainer into cold PBS and cells were pelleted by centrifugation at 350g for 5 min at 4 °C and resuspended in red blood cell lysis buffer, then washed with PBS and pelleted at 350g for 5 min at 4 °C. Lung fibroblasts were seeded on 10cm plates coated with collagen I. The cells were expanded for 5 days in DMEM with 5% P/S and 10% FBS and the medium was replaced after 3 days. 3' end mRNA libraries were prepared for sequencing using a method developed at Dr. Ido Amit's lab, Weizmann Institute of Science 3' RNA-seq for digital gene expression quantitation