Table 3.
Reagents details.
| Antibodies used for immunocytochemistry/flow-cytometry | |||
|---|---|---|---|
| Antibody | Dilution | Company Cat # and RRID | |
| Pluripotency Markers | Mouse anti-SOX2 | 1:200 | Millipore, Cat# MAB4423, RRID:AB_11213224 |
| Pluripotency Markers | PE anti-human SSEA-4 | 1:200 | BioLegend Cat# 330406, RRID:AB_1089206 |
| Pluripotency Markers | Mouse anti-OCT3/4 | 1:200 | Santa Cruz Biotechnology Cat# sc5279, RRID: AB_628051 |
| Pluripotency Markers | Rabbit anti-OCT4 | 1:200 | Abcam Cat #ab19557, RRID:N/A |
| Pluripotency Markers | Mouse anti-Nanog | 1:200 | Millipore Cat# MABD24, RRID:AB_11203826 |
| Differentiation Markers | Rabbit anti-Nestin | 1:200 | Millipore Cat# ABD69, RRID:AB_2744681 |
| Differentiation Markers | Mouse anti-MAP2 | 1:200 | Santa Cruz Biotechnology Cat# sc-74422, RRID:AB_1126216 |
| Differentiation Markers | Mouse anti-alpha-Feto Protein | 1:200 | Millipore Cat# 2004189, RRID: N/A |
| Differentiation Markers | Goat anti-SOX17 | 1:200 | R and D Systems Cat# AF1924, RRID:AB_355060 |
| Differentiation Markers | Goat anti-Brachyury | 1:200 | R and D Systems Cat# AF2085, RRID:AB_2200235 |
| Differentiation Markers | Goat anti-alpha-Muscle Actin | 1:200 | Novus Cat# NB300–978, RRID:AB_2273628 |
| Secondary antibodies | Donkey anti-mouse Alexa Fluor 488 | 1:500 | Thermo Fisher Scientific Cat# A-21202, RRID:AB_141607 |
| Secondary antibodies | Donkey anti-mouse Alexa Fluor 568 | 1:500 | Thermo Fisher Scientific Cat# A10037, RRID:AB_2534013 |
| Secondary antibodies | Donkey anti-rabbit Alexa Fluor 488 | 1:500 | Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792 |
| Secondary antibodies | Donkey anti-goat Alexa Fluor 488 | 1:500 | Thermo Fisher Scientific Cat# A-11055, RRID:AB_2534102 |
| Secondary antibodies | Donkey anti-goat Alexa Fluor 568 | 1:500 | Thermo Fisher Scientific Cat# A-11057, RRID:AB_2534104 |
| Primers | |||
| Target | Forward/Reverse primer (5’−3’) | ||
| Targeted mutation analysis/sequencing | G2019S and 120201 | TTGACCCTTTTTTAAAGCAT/ACAATGTGATGCTTGCATTT | |
| Targeted mutation analysis/sequencing | Y1699C | CACAGGGATTACCGAAAATA/AAGGAATTTTCTTCCTGACAA | |
| Targeted mutation analysis/sequencing | R1441C | TAAAAGGGTGAAGANTTGGG/TTCTCTACCAGCCTACCATG | |
| Targeted mutation analysis/sequencing | N1437H | TTACCCTTGCCTCCAGAATG/AACATTCACCAACATGGAACA | |
| Targeted mutation analysis/sequencing | K1906M | AGGGTATGTGTAGAGGGAAG/CAACGAAAAGACAAACTAGG | |
| Targeted mutation analysis/sequencing | T1348N | CAGTACTCTGAAAGACATGTACA/CCATTCTGGAGGCAAGGGTA | |
| Amplicon Sequencing | 12020T and G20195 | AAGGGACAAAGTGAGCACAGA/CCTGATCACCTACCTGGTGTG | |
| Amplicon Sequencing | Y1699C region | GCTCAACAAGGTTGGGTGTT/TTTTCATATGCCATCTCCCTAA | |
| Amplicon Sequencing | R1441C region | TGGGTTAAGAAAGGCCAAAA/GCCAAAGCATCAGATTCCTC | |
| Amplicon Sequencing | N1437H region | GGATTCTTGCCTGTCGTTTG/TTCCCAATTCAAAATTTTAGTGC | |
| Amplicon Sequencing | K1906M region | GGAAGACGGGAAGGTTAAGG/TTTGCATTTTCACAACTGTATGC | |
| Amplicon Sequencing | T1348N region | TTTGCATTTTCACAACTGTATGC/CATACAGTCTACCAGGTTTCTGGA | |
| CRISPR reagents | |||
| Targeted mutation | crRNA used for Cas9 editing | Donor ago sequence | |
| 12020T | ATTGCAAAGATTGCTGACTA cgg | CCACTCAGCCATGATTATATACCGAGACCTGAAACCCCACAATGTGCTGCTTTTCACACTGTATCCCAATGCTGCCATCATTGCAAAGATTGCTGATTATGGCACTGCTCAGTACTGCTGTAGAATGGGGATAAAAACATCAGAGGGCACACCAGGTAGGTGATCAGGTCTGTCTCATAATTCTATCTTCAGGATGGA | |
| 620195 | ATTGCAAAGATTGCTGACTA cgg | CCACTCAGCCATGATTATATACCGAGACCTGAAACCCCACAATGTGCTGCTTTTCACACTGTATCCCAATGCTGCCATCATTGCAAAGATTGCTGATTATAGCATTGCTCAGTACTGCTGTAGAATGGGGATAAAAACATCAGAGGGCACACCAGGTAGGTGATCAGGTCTGTCTCATAATTCTATCTTCAGGATGGA | |
| Y1699C | TGAAATGCCTTATTTTCCM tgg | AGTGTCCTTTTGCCTTTAGTTTETCTGACCACAGGCCTGTGATAGAGCTTCCCCATTGTGAGAACTCTGAAATTATCATTCGACTATATGAAATGCCTTGTTTTCCAATGGGATTTTGGTCAAGATTAATCAATCGATTACTTGAGATTTCACCTTACATGCTTTCAGGGAGAGGTAAGTATCTAATGAAGACT | |
| R1441C | GGGAAGAAGAAGCGCGAGCC tgg | TTCAAGTGAATGTCACGGAAAGCAAATATCAACAGGAATGTGAGCAGGCCCAGTTTGAAAGCAAACACAAGAGGGTTTTGTGTCTTTCCCTCCAGGCTTGCGCCTCATCTTCCCCTGTGATTCTCGTTGGCACACATTTAGATGTTTCTGATGAGAAGCAACGCAAAGCCTGCATGAGTAAAATCACCAAGGAACTC | |
| N1437H | ACCTTTATATTGAAGAGCCA agg | AGGAATTCTATAGTACTCATCCCCATTTTATGACGCA GCGAGCATTGTACCTTGCTGTCTATGACCTCAGCAAG GGACAGGCTGAAGTTGATGCCATGAAGCCTTGGCTA TTTCATATAAAGGTGATTTGTTCTGATCATTTGAAAA TAGAAAATAATTCATGTGTCTGTGTGCGTGTGTGTGT GTGTGTGTAAGTTA | |
| K1906M | AGCCTATGAAGGAGAAGAAG tgg | ATAATTTATTTAGETTTTTTCGCTTGGACAGCTTAATTTAAGTGCTTTGTATTTTCTTTTCAAAAGGTGATGGCAGTTTTGGATCAGTTTACCGAGCTGCCTACGAAGGAGAAGAAGTGGCTGTGATGATTTTTAATAAACATACATCACTCAGGCTGTTAAGACAAGTAAGAAATTCAATAATATAATTATATTAAAT | |
| T1348N | CATTAATTGCTGCAATAAGG tgg | TTTTTATCTTTCAAATACTAGGTTTCTTCAACAGCGATTAAAAAAGGCTGTGCCTTATAACCGAATGAAACTTATGATCGTGGGAAATACTGGGAGTGGTAAAAACACCTTATTGCAGCAATTAATGAAAACCAAGAAATCAGATCTTGGAATGCAAAGTGCCACAGTTGGCATAGATGTGAAAGACTGGCCTATCC | |
| LRRK2 KO GGAAGACGGGAAGGTTAAGG/TTAAGCTGTCCAAG CGAAAAA | AGCCTATGMGGAGAAGAAG tgg and CATTAATTGCTGCAATAAGG tgg | N/A | |
| Primers | |||
| Target | Forward/Reverse primer (5’−3’) | ||
| Positive internal control primers (Mycoplasma test) | LRRK2 locus | GGAAGACGGGAAGGTTAAGG/TTAAGCTGTCCAAGCGAAAAA | |