Knockdown of RNA-Binding Proteins in NALM-6 Cells

Oligonucleotides with shRNA inserts against eleven RBPs (Supplementary Table 2) were ordered as Ultramer DNA Oligos from Integrated DNA Technologies (Leuven, Belgium). All sequences were based on^{56 (link)}. Oligonucleotides containing shRNA inserts were PCR-amplified with primers 5′-TCTCGAATTCTAGCCCCTTGAAGTCCGAGGCAGTAGGC-3′ and 5′-TGAACTCGAGAAGGTATATTGCTGTTGACAGTGAGCG-3′ and purified with QIAquick PCR Purification Kit (Qiagen). shRNA inserts and miRE18_LT3GEPIR_Ren714 backbone (inducible via Tet-On system) were cut with EcoRI and XhoI (New England Biolabs). Backbone was purified from agarose gel with QIAquick Gel Extraction Kit (Qiagen). The fragments were then ligated with T4 DNA Ligase (New England Biolabs) at 16 °C overnight.
Constructs were transduced into NALM-6 via HEK293T-produced lentiviruses. To this end, 10 cm dishes of HEK293T were transfected using 30 µl Lipofectamine 2000 (Thermo Fisher Scientific) with three plasmids: 4 µg shRNA-producing constructs + 2 µg psPAX2 (lentiviral packaging) + 1 µg pMD2.G (lentiviral envelope) at 72 h prior to transduction. On the first day after transfection, the medium was changed. Work with cells used for lentiviral production was conducted in the S2 laboratory.