GSM8723812: E320K-ELK1-D_Replicate2_Timepoint 1; Homo sapiens; OTHER
Extracted DNA was PCR-amplified with Illumina indexing primers and a second internal primer for sequence alignment. An oligonuclotide pool encoding short linear motifs from the human proteome  with common flanking sequence was amplified by PCR, restriction digested and ligated into the yeast two-hybrid prey plasmid encoding ELK1 between the SpeI and AgeI sites, replacing the native ELK1 D-site. MAX Efficiency DH10B electrocompetent E. coli (ThermoFisher) were transformed with the ligation reaction product. Transformed cells were spread on 15 cm Luria-Bertani Broth (LB) agar plates supplemented with 0.1 mg/ml ampicillin and grown overnight at 37°C. Plasmid DNA was isolated from pooled colonies (Qiagen Maxi prep kit). Colony counts from serial dilution plates confirmed the presence of at least 50 transformants per library sequence. The variable sequence was PCR-amplified and sequenced (Illumina HiSeq 4000) to verify clone representation of all library components. Yeast two hybrid stran PJ694A harboring ERK2 D319N or E320K mutant bait plasmids was transformed with the library plasmid pool for competitive growth Y2H screens.