Soc outgrowth medium

Hdac1 was amplified with flanking AscI (5′-CCCTCACTCGGCGCGatggcgcagacgcagggcaccc-3′) and NsiI (5′-AAGAGGCAGAATGCATGCTCGAGTTAACggccaacttgacctcctcc-3′) restriction sites from the HDAC1 Flag plasmid (gift from Eric Verdin, Addgene plasmid #13820, (39 )) using the KAPA HiFi HotStart ReadyMix (Roche). Using the In-Fusion HD Cloning Kit (Clontech) Hdac1 was cloned into the double digested pRRLs-Avi-MCS-iGFP-BirA (40 (link)) resulting into pRRLs-Avi-HDAC1-iGFP-BirA. Competent GT115 Escherichia coli (Invivogen) were transfected via heat-shock, recovered in SOC Outgrowth Medium (New England Biolabs), plated on LB-Amp plates and incubated overnight. The presence of HDAC1 was tested via colony PCR and sequencing (Eurofins Genomics) (primers: Fwd 5′-CTGCTTCTCGCTTCTGTTCG-3′, Rev 5′-CACACCGGCCTTATTCCAAG-3′).

WT sequences of gfi1b, mCherry, and runx1 +23 enhancer were separately cloned into pJET1.2 vectors using the CloneJet PCR Cloning Kit (Thermo Fisher Scientific). After transformation, the outgrown colonies were selected for DNA isolation. The presence of the insert was confirmed by restriction enzyme digestion using BglII (NEB) followed by agarose gel electrophoresis (1% and 5%). DNA fragments were then used as a polymerase chain reaction template for the Gibson cloning reaction. Fragments were amplified using overhang primers (supplemental Table 1) and purified using a DNA Clean & Concentrator kit (Zymo Research). The pUC19-iTol2 backbone was digested with BamHI-HF (NEB) overnight at 37°C, and NEBuilder HiFi Assembly MasterMix (NEB) was used for the assembly of the fragments. The correct assembly was determined by HindIII restriction enzyme digestion and polymerase chain reaction amplification of the fragments. All transformations were performed using Escherichia coli and by performing heat shock for 30 seconds at 42°C followed by recovery in SOC Outgrowth Medium (NEB) for 1 hour. All colonies were grown in lysogeny broth medium plates supplemented with carbenicillin (50 mg/mL; 1000:1 volume-to-volume ration), and DNA from individual colonies was isolated using a QIAprep Spin Miniprep Kit (Qiagen).