Cv186 lentivirus vector

Human POU6F1 cDNA (1836 bp) and RORA cDNA (1671 bp) were obtained by PCR using the vector LV201-POU6F1 and LV205-RORA (Guangzhou FulenGen Co., Ltd.) and then inserted into CV186 lentivirus vector containing sequence which could express both red fluorescent protein and anti-puromycin protein (Genechem Co., Ltd., Shanghai, China). The POU6F1 PCR fragments and pCMV-HA were double-digested with EcoR I and Xho I and ligated to construct pCMV-HA-POU6F1. Meanwhile, the pCMV-3Tag-1-RORA vector was constructed with BamH I and Hind III. In addition, independent single guide RNAs (sgRNAs) targeting the downstream region of the POU6F1 transcription start site and oligonucleotides specific of shRNAs for RORA were inserted into dCas9-BFP-KRAB (Addgene) and GV298 vector (Shanghai GeneChem Co., Ltd), respectively. The primer sequences were listed in Supplementary Table 5. Stable cell lines were constructed with puromycin for 3–4 weeks.

Human SPI1 cDNA (816 bp, Shanghai GeneChem Co., Ltd, China) were inserted into CV186 lentivirus vector (Genechem Co., Ltd). Human SPIB cDNA (789 bp) construct was provided by Dr. Zhe Liu.⁴⁷ Their truncated fragments obtained using primer sets (Table S5) were inserted into pCMV‐N‐Myc, pCMV‐3Tag‐1C, pGEX‐6P‐1 or pET‐28a (Addgene, Watertown, MA). The shRNAs for SPI1 or SPIB were established by inserting oligonucleotides (Table S6) into GV298 vector (Shanghai GeneChem Co., Ltd), while small interfering RNAs (siRNAs) were synthesized (Table S6).