Table 2.
Reagents details.
| Antibodies and stains used for immunocytochemistry/flow-cytometry | RRID | |||
|---|---|---|---|---|
|
| ||||
| Antibody | Dilution | Company Cat # | ||
|
| ||||
| Pluripotency Markers | Mouse anti-Oct3/4 (C-10) | 1:200 | Santa Cruz #sc-5297 | RRID:AB_628051 |
| Rabbit anti-Nanog (D73G4) | 1:400 | Cell Signaling #4903S | RRID:AB_10559205 | |
| Rabbit anti-Nanog (D73G4) | 1:300 | Cell Signaling #3579S | RRID:AB_2195767 | |
| Rabbit anti-Sox2 (D6D9) | 1:50 | Biolegend #330306 | RRID:AB_1279440 | |
| AF488 anti-human SSEA-3 | 1:400 | Biolegend #330408 | RRID:AB_1089200 | |
| AF647 anti-human SSEA-4 | 1:100 | Biolegend #330614 | RRID:AB_2119064 | |
| AF488 anti-human Tra-1–60 | 1:50 | Biolegend #330706 | RRID:AB_1089242 | |
| AF647 anti-human Tra-1–81 | 1:50 | Biolegend #301910 | RRID:AB_493257 | |
| AF488 anti-human CD15 (SSEA-1) | 1:200 | Biolegend #355210 | RRID:AB_2562013 | |
| Differentiation Markers | PE Mouse anti-human Sox17 | 1:25 | BD #561591 | RRID:AB_10717121 |
| Mouse anti-human FoxA2 | 1:100 | Santa Cruz #sc-101060 | RRID:AB_1124660 | |
| Rabbit anti-FOXG1 | 1:300 | Abcam #196868 | RRID:AB_2892604 | |
| AF647 anti-human PAX6 | 1:20 | BD #562249 | RRID:AB_2644844 | |
| Mouse anti-Hand1 | 1:200 | Novus #NBP2–00576 | RRID:AB_2877685 | |
| CD144 APC | 1:20 | eBioscience #17–1449–42 | RRID:AB_10804754 | |
| Secondary antibodies | Goat anti-mouse IgG2a-AF647 | 1:400 | Jackson Immunoresearch #115–605–206 | RRID:AB_2338917 |
| Goat anti-rabbit IgG-AF488 | 1:400 | Jackson Immunoresearch #111–545–144 | RRID: AB_2338052 | |
| Goat anti-mouse IgG2b-AF488 | 1:400 | Jackson Immunoresearch #115–545–207 | RRID:AB_2338856 | |
| Nuclear stain | Hoechst33342, DAPI | 1 μg/mL | Cell Signalling Technology #4082 | N/A |
| DAPI | N/A | Vector Laboratories | N/A | |
| Site-specific nuclease | ||||
|
| ||||
| Nuclease information | human codon-optimized Cas9 nuclease from S. pyogenes | RRID:Addgene_44719 | Delivery method: Lipofection (Lipofectamine Stem), Enrichment strategy: FACS for GFP expression | |
| BclI-HF restriction endonucleases | New England Biolabs #R3160S | N/A | ||
| Primers and Oligonucleotides used in this study | ||||
|
| ||||
| Episomal plasmids | RRID:Addgene_41824 | gRNA expression vector | ||
| RRID:Addgene_44719 | Cas9-GFP plasmid | |||
| Targeted mutation analysis/Sanger sequencing | ACTG2 exon 8 R257C screen (501 bp region) | GAAGGAGGTTTTCATGGAGATCAA / CTGTTCTGCTTTTCCTGTATCTTG | ||
| Genomic editing | 200 bp ssDNA oligonucleotide repair template with ACTG2 c.769C>T mutation | CCTCCCTGGAGAAGAGCTATGAGCTGCCAGATGGGCAAGTGATCACCATT GGCAATGAGCGCTTCTGCTGCCCTGAGACCCTCTTCCAGCCTTCATTCATA GGTGAGATGCTGCCCACAGTCCCTGCCAATCTCAGGAGGGGAGGGTGGAG GAGTGGGTGAGGTATGGAGAGAGAAACACCAGGAGTCATGGCCACTTTG |
||
| 200 bp ssDNA oligonucleotide repair template with ACTG2 wild-type sequence | CCTCCCTGGAGAAGAGCTATGAGCTGCCAGATGGGCAAGTTATCACCATTG GCAATGAGCGCTTCCGCTGCCCTGAGACCCTCTTCCAGCCTTCATTCATA GGTGAGATGCTGCCCACAGTCCCTGCCAATCTCAGGAGGGGAGGGTGG AGGAGTGGGTGAGGTATGGAGAGAGAAACACCAGGAGTCATGGCCACTTTG |
|||
| gRNA sequence with homology arms targeting ACTG2 locus, used with RRID:Addgene_41824 | TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCAGCCTTCCTTTATTGGTG/ GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCACCAATAAAGGAAGGCTGC | |||
| Genomic target sequence | Genomic location of ACTG2/c.769C>T | chr2:73914835–73914835 | ||
| Genomic location of gRNA binding site | chr2:73914855–73914877 (nucleotide sequence: CCAGCCTTCCTTTATTGGTG AGG) | |||
| Bioinformatic gRNA design tool used | Benchling (2018) | https://www.benchling.com/crispr | ||
| Mycoplasma Detection | 16S Ribosomal RNA (518 bp) | CGCCTGAGTAGTACGTTCGC / GCGGTGTGTACAAGACCCGA | ||
| GAPDH (internal control) (150 bp) | GTGGACCTGACCTGCCGTCT / GGAGGAGTGGGTGTCGCTGT | |||
| Verification of the absence of random plasmid integration events | Kanamycin (RRID:Addgene_41824) (340 bp) | CAGACAATCGGCTGCTCTGA / ATGCGATGTTTCGCTTGGTG | ||
| Ampicillin (RRID:Addgene_44719) (230 bp) | TTGTTGCCGGGAAGCTAGAG / TGATAACACTGCGGCCAACT | |||
| GAPDH (internal control) (150 bp) | GTGGACCTGACCTGCCGTCT / GGAGGAGTGGGTGTCGCTGT | |||