GSM5417951: PRR_iLECs_WT-2; Mus musculus; RNA-Seq
Lungs were harvested at P7, minced and digested in Collagenase-I (Fisher scientific, 17-100-17)/Dispase (Corning, 354235) buffer at 37◦C for 30 min under constant rotation. The digested tissue was triturated ten times through 18G needle to dissociate the clumps and filtered using a 70µm nylon mesh (VWR, 10199-656) to remove any cell debris. The cell suspension was centrifuged at 1200 rpm, 4◦C for 5 min. Single cell suspension were incubated with sheep anti-rat IgG dynabeads (Invitrogen, 11035) coated with CD31 antibody (BD Pharmingen, 553370) at room temperature for 20 min with agitation. Lastly, CD31+ cells were isolated using a magnetic rack separator and RNA was extracted using Genejet RNA purification kit. RNA library construction was performed with the TruSeq RNA Library Prep Kit v2 (Illumina, RS-122-2001) according to the manufacturer instructions. For library preparation, 1 µg of RNA was used for each sample. The resulting mRNA library was quantified using Qubit dsDNA High Sensitivity Assay Kit (Thermofisher, Q32851) and verified using the Bioanalyzer DNA1000 assay kit (Agilent, 5067-1505).