MOLM-13 cells were transfected using Lipofectamine
2000 reagent (Invitrogen) according to the manufacturer’s instructions
using pLKO-TETon-Puro lentiviral vectors expressing shRNAs against
the coding sequence of human METTL3, METTL16, or a scrambled control. Twenty-four hours after spinfection, the
cells were replated in fresh medium containing 1 μg mL–1 of puromycin and kept in selection medium for 7 days. Anti-METTL3
and scrambled shRNAs were induced by treating the cells with 200 ng
mL–1 tetracycline for 3 days, anti-METTL16 was induced
by identical treatment for 2 days.
gRNA assays were performed
using dual gRNA vectors as reported previously.44 (link) Viral supernatants were collected 48 h after transfection.
All transfections and viral collections were performed in 15 cm plates
as mentioned below. For virus production, 5 μg of the above
plasmids and 5 μg of psi-Eco packaging vector were transfected
dropwise into the 293T cells using 47.5 μL TransIT LT1 (Mirus)
and 600 μL of Opti-MEM (Invitrogen). The resulting viral supernatant
was harvested, and transduction of cells was performed in 6-well plates.
After transduction, transduced cells were sorted for BFP (for gRNA).
The gRNA sequences are listed inTable S12 .
2000 reagent (Invitrogen) according to the manufacturer’s instructions
using pLKO-TETon-Puro lentiviral vectors expressing shRNAs against
the coding sequence of human METTL3, METTL16, or a scrambled control. Twenty-four hours after spinfection, the
cells were replated in fresh medium containing 1 μg mL–1 of puromycin and kept in selection medium for 7 days. Anti-METTL3
and scrambled shRNAs were induced by treating the cells with 200 ng
mL–1 tetracycline for 3 days, anti-METTL16 was induced
by identical treatment for 2 days.
gRNA assays were performed
using dual gRNA vectors as reported previously.44 (link) Viral supernatants were collected 48 h after transfection.
All transfections and viral collections were performed in 15 cm plates
as mentioned below. For virus production, 5 μg of the above
plasmids and 5 μg of psi-Eco packaging vector were transfected
dropwise into the 293T cells using 47.5 μL TransIT LT1 (Mirus)
and 600 μL of Opti-MEM (Invitrogen). The resulting viral supernatant
was harvested, and transduction of cells was performed in 6-well plates.
After transduction, transduced cells were sorted for BFP (for gRNA).
The gRNA sequences are listed in
