Effects of the loss of transmembrane adaptor protein WBP1L on bone marrow transplantation efficiency in mouse analyzed using RNA-Seq
Effects of the loss of transmembrane adaptor protein WBP1L on bone marrow transplantation efficiency in mouse analyzed using RNA-Seq
Donor Wbp1l-/- or WT mice were sacrificed by Cervical dislocation. Femurs, tibias and spines were separated, cleaned from muscle tissue, crushed using mortar and pestle and filtered through nylon mesh. Cell suspension was then reconstituted in ACK buffer (150 mM NH4Cl, 0.1 mM EDTA (disodium salt), 1 mM KHCO3) to remove erythrocytes. Next, the bone marrow cells were stained with mix of lineage antibodies (CD3e, B220, Ter119, Gr-1 and CD11b) and separated on autoMACS separator to enrich for immature populations. The sorted unstained fraction was further stained with Lineage antibody cocktail together with KIT, SCA1, Ly5.1, and Ly5.2 antibodies. Ly5.2+Ly5.1- LSK bone marrow cells (Lin-SCA1+KIT+) were then sorted on BDinflux machine. Sorted cells were mixed 1:1 with WT bone marrow competitor cells (Ly5.2+Ly5.1+, extracted using the same procedure as the donor cells) and the mixtures (4x106 cells per recipient) were transplanted into the lethally irradiated (7Gy) Ly5.1+ recipient mice by injection into the tail vein. 22 weeks after transplantation the recipient mice were sacrificed and the donor Ly5.2+Ly5.1- LSK cells were purified. RNA from purified LSK cells was isolated using Rneasy Micro Kit (Qiagen) and treated with DNAse I (Qiagen) for 15 min. Libraries were prepared with a SMARTer® Stranded Total RNA-Seq – Pico Input Mammalian library preparation kit v2 (Takara).