Agilent quikchange 2 site directed mutagenesis kit

We identified putative ESEs with SpliceAid2^{37 (link)}. We used SF3B1 wild-type MEL270 and T47D cells transduced with FLAG-SF3B1 WT or FLAG-SF3B1K700E cDNA (in the backbone of pInducer20 (Addgene, #44012)). After drug selection with Neomycin (Thermo Fisher Scientific, 10131027), the selected cells were treated with 1 μg /ml doxycycline (Sigma, D9891). The BRD9 minigene construct was generated by inserting the DNA fragment containing the BRD9 genomic sequence from exon 14 to exon 15 in between the BamHI and AgeI restriction sites in the FRE5 plasmid (Addgene 62377) via Gibson assembly. BRD9 minigene mutagenesis was performed with the Agilent QuikChange II Site-directed mutagenesis kit with specific primers according to the manufacturer’s directions. For transient transfection experiments, cells were seeded into 24-well plate one day before transfection of BRD9 minigene constructs in the presence of X-tremeGENE HP DNA transfection reagent (Roche) according to the manufacturer’s directions. Forty-eight hours after transfection, cells were harvested and RNA was extracted using Qiagen RNeasy mini kit. Minigene-derived and endogenous BRD9 transcripts were analyzed by RT-PCR using specific primers. Primers and oligonucleotides used in RT-PCR reactions were:

The pBABE-puro plasmid vector containing RelA was constructed by ligating the polymerase chain reaction (PCR)-amplified coding region of murine RelA (amino acids 1 to 549) restricted with EcoRI and SalI. Agilent QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies) was used for all RelA point mutation and deletion variants. DNA binding mutant (RelA^DB) within amino-terminal were mutated by a triple substitution at residues Arg35, Tyr36, and Glu39 to Ala (R35A, Y36A, R39A). Mutagenesis of the carboxyl-terminal region for C-term^Δ, TAD^Δ, and TA1^Δ were performed by displacing residues Pro326, Ala429, and Ser520, respectively, with a STOP codon sequence. All site-specific mutations were verified by Sanger sequencing analysis and sequencing analyses of the resulting RelA mutants showed that the targeted sites were the only changes in the DNA sequence.