Table 1.
Sequencing and cloning oligos
| NGS primers round 1 (5’ -> 3’) | Step | |||
|---|---|---|---|---|
| Handle for round 2 | Primer binding sequence | complete sequence | ||
| NOLC1_NGS_F1 (PD7968) | ACACTCTTTCCCTACACGACGCTCTTCCGATCTC | AATGACGTAACACAGGCCCGC | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCAATGACGTAACACAGGCCCGC | 47, 66 |
| NOLC1_NGS_F2 (PD7969) | ACACTCTTTCCCTACACGACGCTCTTCCGATCTAC | AATGACGTAACACAGGCCCGC | ACACTCTTTCCCTACACGACGCTCTTCCGATCTACAATGACGTAACACAGGCCCGC | 47, 66 |
| NOLC1_NGS_F3 (PD7970) | ACACTCTTTCCCTACACGACGCTCTTCCGATCTGAC | AATGACGTAACACAGGCCCGC | ACACTCTTTCCCTACACGACGCTCTTCCGATCTGACAATGACGTAACACAGGCCCGC | 47, 66 |
| NOLC1_NGS_F4 (PD7971) | ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGAC | AATGACGTAACACAGGCCCGC | ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACAATGACGTAACACAGGCCCGC | 47, 66 |
| NOLC1_NGS_R (PD7972) | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | CGAGCACGAGGGGATACAGGTC | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGAGCACGAGGGGATACAGGTC | 47, 66 |
| pDY0181_GFP_insert_NGS_R (PD6893) | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | GAACTCCACGCCGTTCAGGG | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAACTCCACGCCGTTCAGGG | 47, 66 |
| ACTB_NGS_F1 (PD0966) | ACACTCTTTCCCTACACGACGCTCTTCCGATCT | CCGACCTCGGCTCACAGCG | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTGGCGGCCTAAGGACTCG | 47, 66 |
| ACTB_NGS_F2 (PD0967) | ACACTCTTTCCCTACACGACGCTCTTCCGATCTA | CCGACCTCGGCTCACAGCG | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTGGCGGCCTAAGGACTCG | 47, 66 |
| ACTB_NGS_F3 (PD0968) | ACACTCTTTCCCTACACGACGCTCTTCCGATCTGA | CCGACCTCGGCTCACAGCG | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTGGCGGCCTAAGGACTCG | 47, 66 |
| ACTB_NGS_F4 (PD0969) | ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGA | CCGACCTCGGCTCACAGCG | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTGGCGG | 47, 66 |
| CCTAAGGACTCG | ||||
| ACTB_NGS_F5 (PD0970) | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGA | CCGACCTCGGCTCACAGCG | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTGGCGGCCTAAGGACTCG | 47, 66 |
| ACTB_NGS_F6 (PD0971) | ACACTCTTTCCCTACACGACGCTCTTCCGATCTACTGA | CCGACCTCGGCTCACAGCG | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTGGCGGCCTAAGGACTCG | 47, 66 |
| ACTB_NGS_F7 (PD0972) | ACACTCTTTCCCTACACGACGCTCTTCCGATCTTACTGA | CCGACCTCGGCTCACAGCG | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTGGCGGCCTAAGGACTCG | 47, 66 |
| ACTB_NGS_F8 (PD0973) | ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTACTGA | CCGACCTCGGCTCACAGCG | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTGGCGGCCTAAGGACTCG | 47, 66 |
| ACTB_NGS_R (FP0952) | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | CCACCCAGCCAGCTCCC | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCACCCAGCCAGCTCCC | 47, 66 |
| ddPCR primers and probe (5’ -> 3’) | ||||
| NOLC1_ddPCR_F (PD1022) | TGGAGCCCACCCTTTCCGT | 84 | ||
| ACTB_ddPCR_F (PD2053) | CCCGGCTTCCTTTGTCC | 84 | ||
| attL_ddPCR_probe (PD1219) | /56-FAM/CC GGC TTG T/ZEN/C GAC GAC GGC G/3IABkFQ/ | 84 | ||
| Cloning primers (5’ -> 3’) | ||||
| Payload-Gibson-F | ACCACCGCGGTCTCAGTGGTGTACGGTACAAACCCAGCTACCGGTNNNNNNNNNNNNN | 24 | ||
| Payload-Gibson-R | AATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCTAGATCANNNNNNNNNNNNN | 24 | ||
| Nicking guide PCR F (replace N with spacer sequence) | ATggtctcgcaccgNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaaaataaggct | 13 | ||
| Nicking guide PCR R (universal) | atggtctctaaaagcaccgactcggtgccac | 13 | ||
| atgRNA (5’ -> 3’) | 5’ overhang | atgRNA sequence | 3’ overhang | |
| pDY2259 hNOLC1 atgRNA Paired Guide 1 (Addgene ID: 220989) | tcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttc | GCGTATTGCCTGGAGGATGGgttttagagctagaaatagcaagttaa | tttttttaagcttgggccgctcgaggtacctctctacatatgac | 37, 64 |
| ttggctttatatatcttgtggaaaggacgaaacacc | aataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcccggatgatcctgacgacggagaccgccgtcgtcgacaagccggcctcctccagg | atgtgagcaaaaggccag | ||
| pDY2260 hNOLC1 atgRNA Paired Guide 2 (Addgene ID: 220990) | tcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacacc | gTATTGGCCACCTCTGAGAGTgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcggccggcttgtcgacgacggcggtctccgtcgtcaggatcatccggctcagaggt | tttttttaagcttgggccgctcgaggtacctctctacatatgacatgtgagcaaaaggccag | 37, 64 |
| pDY2261 hACTB atgRNA Paired Guide 1 (Addgene ID: 220991) | tcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacacc | GCTATTCTCGCAGCTCACCAgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcATGATCCTGACGACGGAGACCGCCGTCGTCGACAAGCCtgagctgcgagaa | tttttttaagcttgggccgctcgaggtacctctctacatatgacatgtgagcaaaaggccag | 37, 64 |
| pDY2262 hACTB atgRNA Paired Guide 2 (Addgene ID: 220992) | tcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacacc | GGAGGGGAAGACGGCCCGGGgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcggcttgtcgacgacggcggtctccgtcgtcaggatcatgggccgtcttccc | tttttttaagcttgggccgctcgaggtacctctctacatatgacatgtgagcaaaaggccag | 37, 64 |
| Sanger sequencing primers (5’ -> 3’) | ||||
| hU6-F | GAGGGCCTATTTCCCATGATT | 12 | ||
| LKO.1 5’ | GACTATCATATGCTTACCGT | 12 | ||
| pCI-rev | GCAATAGCATCACAAATTTCAC | 29 | ||
| Synthetic RNA sequences | ||||
| mNOLC1 twinPE pegRNA 1 | mA*mG*mU*UAAGGAGGCGAGGGCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGCUUGUCGACGACGGCGGUCUCCGUCGUCAGGAUCAUCCCUCGCmC*mU*mC | 37, 64 | ||
| mNOLC1 twinPE pegRNA 2 | mA*mC*mA*CCGAGACCUCCAGCCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCAUGAUCCUGACGACGGAGACCGCCGUCGUCGACAAGCCGCUGGAGmG*mU*mC | 37, 64 | ||
| IVT primers (5’ -> 3’) | ||||
| PASTE IVT F (PD3771) | agataTAATACGACTCACTATAGGGAGAgccgccaccatgaaacgga | Box 3 | ||
| PASTE IVT R (PD3762) | cga ggc tga tca gcg ggt tta aac | Box 3 |