NextSeq 500 sequencing; Bulk RNA-seq evaluation of in vitro cultured Sox9+ mouse lung progenitors isolated from E12.5 embryonic lung progenitors
Bulk RNA-seq evaluation of in vitro cultured Sox9+ mouse lung progenitors isolated from E12.5 embryonic lung progenitors
Isolation of E12.5 mouse embryonic lung progenitors: E12.5 mouse embryos were collected from pregnant Sox9GFP females. E12.5 embryonic lungs were carefully dissected with tungsten forceps. The heart and esophagus were carefully removed and lungs were pooled (typically 7 to 9 lungs per litter). E12.5 lungs were incubated in 5 mL of dissociation buffer (Collagenase I 100U/mL, Collagenase II 100U/mL, DNase 100U/mL and Trypsin 50U/mL in phenol red-free Leibovitz L-15 basal media) for 20 minutes with frequent trituration until the embryonic lungs were fully dissociated. Dissociated cells were washed with an equal volume of collection buffer (20% serum in Leibovitz L-15) and filtered through a 100 µm cell strainer. Cells were then centrifuged for 10 minutes at 2000 rpm (4 oC). The pellet was re-suspended in pre-warmed (37 oC) suspension buffer (2% serum, HEPES 25 mM, EDTA 2 mM in Leibovitz L-15) and filtered again through a 40 µm cell strainer. After washing in FACS buffer (2%Serum + 0.2% bovine serum albumin [BSA] in Leibovitz L-15) and centrifugation for 5 minutes at 2000 rpm (4 oC), cells were blocked for 10 minutes at room temperature (RT) in FACS buffer before 30 mins of incubation with anti-Cdh1, anti-Pecam1/CD31, anti-Ptprc/CD45 and anti-Ter119 eFluor-conjugated primary antibodies at 4oC. For adult lung samples, anti-Cdh1 or anti-EpCAM antibodies were used only. After antibody incubation, cells were washed in FACS buffer and pelleted before being resuspended in 500 µL of suspension buffer + DAPI (0.1 µg/mL). Sorting was performed on a BD FACS Aria II cytometer. Relevant cell fractions were collected and followed by RNA extraction. Primary Sox9+ lung progenitor cell culture and expansion: Typically, 1000 Sox9GFP+Cdh1+Pecam1-Ptprc-Ter119- sorted cells were embedded in 40 µL of Growth Factor Reduced, Phenol Red-Free Matrigel (10mg/mL, Corning #356231), re-suspended on ice and seeded as a single droplet in 24-well plates. The cell-containing Matrigel droplet was allowed to solidify for 20 minutes at 37 oC before 500 µL of LPM-3D media (Fgf10 50ng/ml; Fgf9 50ng/ml; Egf 50ng/ml; CHIR99021 3µM; BIRB796 1µM; Y27632 10µM; A8301 1µM; Heparin 5µg/ml; Insulin 10µg/ml; Transferrin 15µg/ml) was added. Full media was changed every 2 days and cells were passaged every 10 to 12 days with a 1:8 to 1:10 split ratio. For passaging, the Matrigel droplet was incubated with a mixture of Dispase II (Thermofisher #17105041), Collagenase IV (Thermofisher #17104019) and Papain (Worthington #LS003118) (0.5 mg/mL each) at 37oC and triturated for 30-60 minutes until the Matrigel was fully digested and colonies were dissociated into single cells. Cells were then washed with DMEM/F12 and centrifuged for 10 minutes at 2000rpm (4 oC); the supernatant was then removed and cells were re-embedded in Matrigel as described above. Sorting was performed on a BD FACS Aria II cytometer. Sox9GFP+ cell fractions were collected and followed by RNA extraction. Smart-Seq2. Total RNAs (1ng) from 1x105 FACS-sorted cells with RIN value >7 were further processed to synthesize cDNAs according to Picelli et al, 2014 Smart-seq2 protocol. cDNAs size distribution and concentration was assessed by Agilent Bioanalyzer using High sensitivity DNA analysis kit (Agilent technologies #5067-4626). Libraries were constructed using the Illumina Nextera XT DNA Sample Preparation kit (Illumina #FC-131-1096). Libraries were quantified by using KAPA Library Quantification Kit (Kapa Biosystems #KK4854) and size distribution was assessed by Agilent Bioanalyzer. RNA-seq libraries were sequenced on Illumina NEXTSeq platform (single-end 76bp).
Experimental Factor: developmental stage
adult
Experimental Factor: phenotype
EpCAM+
Experimental Factor: passage
not applicable