title: GSM5098836: Ahnak_KO_TGFb_minus; Mus musculus; RNA-Seq
library_construction_protocol: Mouse pulmonary endothelial cells were isolated from the lungs of 6 to 8-wk-old WT or Ahnak-/- mice. Each mouse was sacrificed by CO2 inhalation. After reperfusion with PBS, the lungs were removed and digested in DMEM supplemented with 0.1% type I collagenase (Worthington) for 60 minutes at 37C. A total of 15 mL of collagenase solution was used per mouse, and the mixture was shaken vigorously during digestion before being titrated into the fixed cannula three times. The collagenase solution containing cells was then strained through a 70-μm strainer. The filtered cell suspension was centrifuged at 1300 rpm for 8 min. After removal of the supernatant, the cell pellet was resuspended twice in 20 mL of 10% DMEM and again centrifuged at 1300 rpm for 8 min. After the supernatant was removed, the cell pellet was resuspended in 4 mL of 10% DMEM, and Dynabeads (Invitrogen) coupled to an anti-ICAM-2 antibody were added to the cells. The Dynabeads/cell mixture was incubated for 30 minutes at 4C. The bead-bound cells were collected using a magnet, washed 5 times in 10% DMEM and once in serum-free DMEM, and digested for 8 minutes at 37C in 1 mL of trypsin/EDTA to release the beads from the cells. The bead-free cells in 10% DMEM were centrifuged, resuspended in 4 mL of EGM-2 culture medium (Lonza), plated onto a gelatin-coated 60mm culture dish, and left undisturbed for approximately 4-5 days. cDNA libraries were constructed with the TruSeq mRNA library kit using 1 μg of total RNA. Paired end sequencing of 100-mers length was performed using an Illumina HiSeq4000 system.