GSE130781
Transcriptome (RNA-Seq), translatome (Ribo-Seq), and YB-1/YB-3-bound mRNAs (RIP-Seq) of HEK293T, HEK293TdeltaYB-1, and HEK293T+HA-YB-1 cells
We present results of RNA-Seq, Ribo-Seq, and RIP-Seq (YB-1, YB-3) experiments performed in HEK293T cells, as well as in HEK293T cells with YB-1 knockout and overexpression. The data shows YB-1 function as a global translation inhibitor and YB-3 ability to substitute YB-1 in its function in YB-1 knockout mutant. Overall design: Cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg streptomycin. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 and passaged by standard methods. YB-1 knockout was performed with CRISPR/Cas9. YB-1 overexpression was achieved by transit transfection of pcDNA3.1 HA-YB1 plasmid. RIP-Seq was performed using (4202, CST; A303-230A, Bethyl) antibodies for YB-1 and (A303-070A, Bethyl) antibodies for YB-3. Libraries for Ribo-Seq were prepared with TruSeq Ribo Profile Kit (Illumina). The same kit was used for size-matched RNA-Seq (used as the control for Ribo-Seq). RIP-Seq and its control RNA-Seq were produced with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB). Ribosomal RNA depletion was performed with Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat)(Illumina) for Ribo-Seq and with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) or NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB) for RIP-Seq. Libraries were sequenced on Illumina HiSeq 2000 and Illumina NextSeq 500.