Lenti-miR-Off-GFP-hsa-miR-15b vector (anti-miR-15b), Lenti-miR-Off-GFP-hsa-miR-219-5p vector (anti-miR-219-5p) and pLenti-III-GFP-mir-Off control vector (ABM Inc., Canada) were purchased from ABM Incorporation. Bacterial colonies were grown in LB-ampicillin broth by incubation with shaking at 37 °C overnight. Then plasmids were extracted using the DNA purification kit (DNA-spin, iNtRON Biotechnology, Seoul, Korea).
Nucleofection of CD34+ HSCs was performed according to the manufacturer's instructions with the nucleofector machine (Lonza). Cells (8×105) were resuspended in 100 μL of HSCs nucleofection solution (Lonza), and 5 μg of anti hsa-miR-15b, 5 μg of anti miR-219-5b, and 5 μg of control scramble vector (abm Inc., Canada) was added. Samples were transferred into certified cuvettes (Lonza) and transfected by nucleofection with program U-008. Fresh medium (500 μL) was added immediately after transfection to each cuvette, and the cells were plated and incubated at 37 °C for 10 days. After nucleofection, HSCs were stained with 0.4 % Trypan Blue (vital dye) (Sigma-Aldrich) for 1 min at room temperature and the number of live HSCs (unstained) were counted every 48 hours using a hemocytometer.
Nucleofection of CD34+ HSCs was performed according to the manufacturer's instructions with the nucleofector machine (Lonza). Cells (8×105) were resuspended in 100 μL of HSCs nucleofection solution (Lonza), and 5 μg of anti hsa-miR-15b, 5 μg of anti miR-219-5b, and 5 μg of control scramble vector (abm Inc., Canada) was added. Samples were transferred into certified cuvettes (Lonza) and transfected by nucleofection with program U-008. Fresh medium (500 μL) was added immediately after transfection to each cuvette, and the cells were plated and incubated at 37 °C for 10 days. After nucleofection, HSCs were stained with 0.4 % Trypan Blue (vital dye) (Sigma-Aldrich) for 1 min at room temperature and the number of live HSCs (unstained) were counted every 48 hours using a hemocytometer.