Plh spsgrna2

To activate the 3′RR IgH enhancer, we exploited the CRISPRa technology (40 (link)). The sgRNA (hs1.2 and hs4) sequence was obtained from previously published report (61 (link)). Briefly, oligonucleotides were annealed in the following reaction: 10 μM guide sequence oligo, 10 μM reverse complement oligo, and 50 mM NaCl in tris-EDTA buffer with the cycling parameters of 95°C for 2 min and then ramp down to 25°C at 5°C/min. The annealed oligos were cloned into the sgRNA vector (pLH-spsgRNA2, Addgene, 64114) using a Golden Gate Assembly strategy including 100 ng of circular sgRNA vector plasmid, 0.2 μM annealed oligos, 20 U of Bbs I restriction enzyme, 750 U of T7 DNA ligase (Enzymatics, L6020L), and 1× T7 ligase buffer with the cycling parameters of 37°C for 5 min, followed by 60°C for 5 min. Insertion of sgRNA was validated by Sanger sequencing (Genetic Analyzer, 3130xl). The pcDNA-dCas9-p300^C was purchased from Addgene (no. 61357). Briefly, 500 ng of dCas9-p300^C expression vector and 500 ng of individual gRNA expression vectors were transfected in Med12^KD cells, and cells were incubated for 48 hours. After the incubation, the cells were CIT (+)–stimulated for another 48 hours before collecting the cells used for subsequent analysis.