GSM7844309: Pdgfra_SmoM2_Replicate3; Mus musculus; RNA-Seq
Ducts were microdissected and placed in 1 ml of HBSS with 1.8 mM Ca, 0.8 mM Mg, 1 mg dispase (Roche, 04942078001), 1 mg pronase (Sigma, P5147), and 1mg Collagense IV (Gibco, 17104019) and cut into small pieces with microdissecting scissors. These were incubated for 20 minutes at 37oC on a rocker with continued mincing of the tissue every 5 minutes. 500 ul of ice cold 1x PBS, 5mM EDTA, and 0.5% BSA was added and tube was placed in a 4C rocker for 5 minutes with large bore pipet titration to break up any clumps. This was filtered through a 100 μm filter. This was was rinsed twice with 1x PBS, 5mM EDTA, and 0.5% BSA prior to flow cytometry. ~1000 mesenchymal cells per individual animal bile duct were sorted into lysis buffer where after the Smartseq3 protocol with the Smartseq2 TSO and ISPCR primers was used to generate full length cDNA libraries. Libraries for Illumina sequencing were generated by using New England Biolabs Next Ultra II FS DNA (E7805L).