Table 2.
Reagents details.
| Antibodies and stains used for immunocytochemistry/flow-cytometry | |||
|---|---|---|---|
| Antibody | Dilution | Company Cat # and RRID | |
|
| |||
| Pluripotency Markers | Rabbit anti-OCT4 | 1:100 for immunostaining 1:60 for flow cytometry |
Abcam Cat# ab181557, RRID: AB_2687916 |
| Rabbit anti-SOX2 | 1: 100 | Abcam Cat# ab92494, RRID: AB_10585428 | |
| Rabbit anti-NANOG | 1: 100 | Abcam Cat# ab21624, RRID: AB_446437 | |
| Differentiation Markers | Rabbit anti-PAX6 | 1:100 | BioLegend Cat# PRB-278P, RRID: AB_291612 |
| Rabbit anti- BRACHYURY | 1:100 | Abcam Cat# ab209665, RRID: AB_2750925 | |
| Rabbit anti-FOXA2 | 1:100 | Abcam Cat# ab108422, RRID: AB_11157157 | |
| RPE Markers | Rabbit anti-ZO-1 | 1:100 | Thermo Fisher Scientific Cat# 402200, RRID: AB_2533456 |
| Mouse anti-MITF | 1:100 | Abcam Cat# ab3201, RRID: AB_303601 | |
| Mouse anti-RPE65 | 1:100 | Novus Biologicals Cat# NB100-35 RRID: | |
| AB_10002148 | |||
| Western Blot analysis | Rabbit anti-LRP2 | 1:300 | Proteintech Cat# 9700-1-AP, RRID: N/A |
| Secondary antibodies | Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H + L) | 1:1000 | Yeasen Cat# 34212ES60, RRID: N/A |
| AlexaFlour488 goat anti-rabbit IgG | 1:1000 | Thermo Fisher Scientific Cat# A-11008, RRID: AB_143165 | |
| AlexaFlour555 goat anti-mouse IgG | 1:1000 | Thermo Fisher Scientific Cat#A-21422, RRID: AB_2535844 | |
| Peroxidase-Conjugated Goat Anti-Rabbit IgG (H + L) | 1:5000 | Yeasen Cat# 33101ES60, RRID: N/A | |
| Nuclear stain | DAPI | 1:1000 | Abcam Cat# ab104139, RRID: N/A |
| Site-specific nuclease Nuclease information | N/A | N/A | |
| Delivery method | |||
| Selection/enrichment strategy | FACS | FACS | |
| Primers and Oligonucleotides used in this study | |||
| Target | Forward/Reverse primer (5′–3′) | ||
| Targeted mutation analysis/sequencing | LRP2, 2nd Exon of common sequence of three | GCAGTATCTGGAGAATCTCTGTTTG/ | |
| transcripts | GAGTTTCCACTAAATCTTGTCATTCAGC | ||
| gRNA oligonucleotide/crRNA sequence | LRP2 | GGCCAAGCCATTGGGCCATCC | |
| Potential random integration-detecting PCRs | PB1 | ATACGATGTTCCAGATTACGCT/ GGTGTTTCGTCCTTTCCACAAG | |
| PB2 | CGTGTTTATCTCGTCAACTTGTTGG/CCAGTTTGGAACAAGAGTCCACTAT | ||
| PB3 | CTCGAGGCGTTGACATTGAT/GTGGCACCGGTAGTTAGCC | ||
| Top off-target mutagenesis predicted site sequencing | POT1 | CTTTGCCCGGCCAAGAATTC/TCTGTCAGGCATCATGCTGGG | |
| POT2 | GTGGCATTCCGAATTCTGGC/GTGTTCACACCAGAGCCTGAG | ||
| POT3 | CAGATGGTCAGAGCAGGCTC/TGATGAACCCTTGGGCCAAAG | ||
| POT4 | CAGACGTGCCGATGAAGAGA/CCACAGGAACACTAGGCCAGT | ||
| POT5 | AGTTTACTTGGTGTTCATTACCCA/TGGGCATTTGGAGGGACATCG | ||