design: Total RNA-seq upon degradation of OGT by siRNA
library_construction_protocol: Growing medium was changed 12h after the experiment and the cells collected 48h after siRNA transfection. Cells were resuspended in PBS and centrifuged at 200g for 5 min  E14 WT ES cell lines were grown in Serum/LIF and 2i media without penicillin streptavidin on a 6 cm plate surface. Before cell transfection, 5 μl of Lipofectamine2000 (Invitrogen #11668-019) were mixed with 250 μl of Opti MEM reduced Serum Medium (Gibco #51985-034) and incubated for 5 min at RT.  10 μl of 10 μM OGT siRNA (Santa Cruz Biotechnology #sc-40781) were mixed with 250 μl of Opti-MEM and then combined with the Lipofectamine2000-Opti-MEM mix to make a transfection mix and incubated for 20 min at RT. Cells were detached with TrypLE and counted to plate 300 x105 cells/well. 500 μl of transfection mixture were mixed with the cell suspension and the solution was transferred on a pre-gelatinized 6 cm plate. In parallel, a negative control was prepared following the same conditions but using a non-targeting siRNA (Santa Cruz Biotechnology #sc-37007), and a negative control reaction without siRNA was also processed. Cells were centrifuged at 0.8 rcf for 4 mins, washed in PBS and resuspended in 1ml TRIzol reagent (Invitrogen) per cell pellet and the cell lysate was stored at -80℃. For RNA-seq of OGT Knockdown wild-type ES cells, cells were lysed for RNA collection as insection 2.10. DNase treatment and RNA extraction was done as in 2.10. Samples containing 1 μgtotal RNA as the starting concentration were used. rRNA depletion was conducted using theNEBNext rRNA Depletion Kit v2 (NEB #E7400L) and sample libraries were prepared using theNEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760S). Quality ofcDNA generated was tested on a TapeStation.