Ecori and xhoi

Hot fusion primer pairs were created using CE Design V1.04. Each primer contained a 17–30 bp sequence from the pIRES-hrGFP-1a vector and a gene-specific sequence fragment.
F-primer: agcccgggcggatccgaattcATGCCCGAGATAGTGGATACCTG
R-primer: gtcatccttgtagtcctcgagATCCACTTTTAAGTCTTTCCCCAC.
At 37 °C for 2 to 3 h, we digested the pIRES-hrGFP-1a vector with EcoRI and XhoI (NEB). The enzyme-digested vector was purified on a Qiagen column kit using 1.0% agarose gel. HeLa cells’ total RNA was obtained using Trizol. Oligo dT primers were used to transcribe the purified RNA for cDNA. Following that, PCR amplification was used to synthesize the inserted fragment. PCR insert and linearized vector digested with EcoRI and XhoI (NEB) were combined in a PCR microtube and ligated with Vazyme’s ClonExpress® II One Step Cloning Kit (Vazyme, Nanjing, Jiangsu, China). Chemical transformation was used to introduce plasmids into Escherichia coli strains. We incubated cells overnight at 37 °C on LB agar plates containing 1uL/ml ampicillin. Finally, 28 cycles of colony PCR were performed on the backbone vectors using universal primers to screen colonies.

We employed CE Design V1.04 (Vazyme, Nanjing, China) to design the primer pairs utilized for Hot Fusion. Every primer includes gene-distinct fragment sequence and a 17-30 bp pIRES-hrGFP-1a vector sequence.
F-primer: agcccgggcggatccgaattcATGCAGGATGATTTACTGATG R-primer: gtcatccttgtagtcctcgagGCTCCAGCGGAACGGGAC Using EcoRI and XhoI (NEB), we digested the pIRES-hrGFP-1a vector at 37° C for 2h~3h. Then, the vector digested by the enzyme gel-electrophoresed on 1.0% agarose gel, followed by purification employing the Qiagen column kit (Qiagen. Inc, Valencia, CA). Using Trizol reagent (15596, Life Technologies, USA), we extracted total RNA from the HT22 cells. Subsequently, we reverse-transcribed the RNA to cDNA using an oligo-dT primer. After that, the insert fragment using PCR, we detected the insert fragment. Ligation of the vector that was linearized using digestion with EcoRI and XhoI (NEB) and the PCR insert was accomplished using ClonExpress® II One Step Cloning Kit (Vazyme). Through chemical transformation, we introduced the plasmids into the Escherichia coli strain. Subsequently, the cells were grown on LB agar plates added gL/ml ampicillin in an overnight incubation at 37° C. We screened the colonies through colony PCR (28 cycles) using universal primers (positioned on the backbone vector). The inserts sequence were Sanger sequenced for verification.

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.