BioSample

Jurkat cells (a human T lymphocyte cell line, RCB0806) were obtained from the RIKEN BRC. dCas9-VP64_GFP (plasmid 61422; Addgene) and Lenti_MS2_P65_HSF1_2A_puro (plasmid 118699; Addgene) were gifts from F. Zhang and C. Vakoc, respectively. CRISPRa- Jurkat cell line stably expressing dCas9-VP64 and MS2_P65_HSF1 was established by lentiviral transduction and subsequent selection of GFP-expressing puromycin-resistant cells. Protospacer adjacent motif (PAM) sequence for each bidirectionally transcribed candidate enhancer (btcEnh) region was designed using Custom Alt-RTM CRISPR-Cas9 guide RNA design tool. A 130-base single-guide RNA (sgRNA) harboring an MS2 stem-loop sequence was synthesized by IDT with 2??-O-methyl-modified phosphorothioate modifications at three bases at each 5?? and 3?? end. Non-targeting control sgRNA or targeting sgRNA (100 nM) was transfected to CRISPRa-Jurkat cells using DharmaFECT 4 (Horizon Discovery).