Pspcas9 2a puro px459

To generate the CRISPRa cell line, the CRISPR/Cas9 and nonhomologous end joining (NHEJ) pathway (CRISPR/Cas9-NHEJ)-mediated genome editing strategy was applied [34 (link)]. First, the CRISPR/Cas9 plasmids, pSpCas9 2A-Puro (PX459) (a gift from Feng Zhang, Addgene plasmid #62988) targeting the 3′ region of chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (GAPDH #1) and targeting the common region of the three CRISPRa vectors (CRISPRa #1), Cas9m4-VP64 (Addgene plasmid #47316) [35 (link)], SP-dCas9-VPR (Addgene plasmid #63798) [13 (link)], or pcDNA-dCas9-p300 Core (Addgene plasmid #61357) were constructed [35 (link),36 (link)]. The 2.5 × 10⁵ DF-1 cells were then transfected with 1 µg of GAPDH #1, 1 µg of CRISPRa #1, and 2 µg of each CRISPRa vector using Lipofectamine 3000 according to the manufacturer’s instructions. The transfected cells were treated with Geneticin Selective Antibiotic (G418, 300 µg/mL) (Thermo Fisher Scientific, Waltham, MA, USA, 24–48 h post transfection, and the drug selection was maintained to establish cell lines for at least 2 weeks. The gRNA and oligo sequences used in all-in-one CRISPR/Cas9 vector construction are listed in Table S1.

4T1 or EO771 cells were transduced with Pspcas9-2A-puro-px459 (Addgene 48139) constructs containing each of Padi4 single-guide RNAs designed by F. Zhang’s laboratory, which was followed by 2 μg ml⁻¹ puromycin selection for 2 d. Immunoblotting and genotyping were used for verifying gene knockout. Primers are listed in Supplementary Table 3.
For Padi4 knockdown, the 4T1 cells were transduced with lentiviral particles in 8 μg ml⁻¹ of polybrene for 5 h. After 1 d, cells were selected by 2 μg ml⁻¹ of puromycin for 3 d. For Padi4 knockdown, Mission shRNAs (Sigma, TRCN0000101831 and TRCN000101833) were used.