title: GSM9344682: P5 Smad4 Mut 2; Mus musculus; RNA-Seq
library_construction_protocol: P5 lungs from Smad4 f/f and Smad4 f/f;Cdh5-CreERT2 pups were dissected, minced, and digested in collagenase I (Thermo Fisher Scientific,17-100-17)/dispase (Corning, 354235) buffer at 37·C for 30 minutes under constant rotation. The digested tissue was triturated 10 times through an 18G needle to dissociate the clumps and filtered using 70 µm nylon mesh (VWR, 10199-656) to remove any cell debris. The cell suspension was centrifuged at 250g at 4°C for 5 minutes. Single-cell suspensions were incubated with sheep anti–rat IgG dynabeads (Invitrogen,11035) coated with CD31 antibody (BD Pharmingen, 553370) at room temperature for 20 minutes with agitation. CD31+ cells were isolated using a magnetic rack separator and quantified using Qubit RNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32852). RNA integrity was determined using the Bioanalyzer RNA 6000 Nano assay kit (Agilent, 5067-1511). RNA (0.5 μg per sample) was used for RNA library construction with a TruSeq RNA Library Prep Kit v2 (Illumina, RS-122-2001) as per the manufacturer'r instructions; independent biological samples from HHTf/f and HHTiECKO mice were used to create individual RNA-Seq libraries. The quality and quantity of mRNA library was determined by Qubit dsDNA High Sensitivity Assay Kit (Thermo, Q32851) and Bioanalyzer DNA1000 assay kit (Agilent, 5067-1505), respectively.