Illumina HiSeq 2500 paired end sequencing; RNA-seq investigating mouse derived and iPSC derived macrophage populations before and after pulmonary transplantation
RNA-seq investigating mouse derived and iPSC derived macrophage populations before and after pulmonary transplantation
iPSC: iPSCs were derived from lineage negative bone marrow cells of a C57Bl/6 Ly5.1 mouse using a lentiviral vector expressing the 4 yamanaka factors Oct4, Sox2, Klf4 and c-Myc under an SFFV promoter at an MOI of 10. The clone 10.3 was then expanded on a feeder layer of C3H murine embryonic fibroblasts in presence of LIF to maintain their undifferentiated state. For RNA collection cells were first depleted of feeder cells by cultivating in absence of MEF for 2 days and then RNA was isolated.  BM-Mph: macrophages were differentiated from bone marrow of C57Bl/6 Ly5.1 donor animals. Bone marrow was flushed out from femurs and tibias and depleted of the red cells. After depletion cells were differentiated for 5-7 days in RPMI medium supplemented of 10% serum, 1% L-glu, 1% P/S and 30% L929 supernatant containing M-CSF.  AMph: lungs of C57Bl/6 Ly5.1 animals were flushed with 1ml PBS 3 times using a 27G cannula to retrieve the alveolar macrophages. after flushing the cells were further purified by sorting for the surface marker SIGLEC-F iPSC-Mph: the same iPSCs described above were differentiated to macrophages. First cells were pre-cultivated in absence of MEF feeder cells for 2 days and then were allowed to form embryoid bodies in a 10cm dish for 7 days on a shaker. 5 days into EB formation the medium was supplemented with IL-3 and SCF to induce hematopoietic differentiation. EBs were then dissociated in collagenase IV for 45 minutes resuspending the bodies every 15 minutes. CD41 positive cells were isolated by FACS and further differentiated in macrophages for 7-10 days in RPMI supplemented with 30% L929 supernatant containing M-CSF. PMT-Mph: iPSC-Mph were administered via the trachea using a cannula into anaesthetise animals in a volume of 70ul PBS per animal. After 8 weeks from transplantation the animals were sacrificed and CD45.1 positive cells were isolated by FACS from BALF and lungs and used for RNA isolation.   RNA was isolated using the RNAeasy Plus Micro Kit (QIAGEN). Total RNA (10ng) was amplified using the Ovation RNA-Seq System v2 (NuGEN) according to the manufacturer's protocol The libraries were prepared with the Nextera XT DNA Sample Preparation kit (Illumina Technologies) and the concentrations were measured using the Qubit dsDNA HS assay