| Step | Name | Sequence (5’–>3’) | Notes |
|---|---|---|---|
| 3.1 | CROPseq-PrimeF-BgL-BsmBI | GAGCCTCGTCTCCCACCGNNNNNNNNNNNNNNNNNNNNGTTTTGAGACGCATGCTGCA | The N20 sequence is a random string of oligonucleotides |
| 3.1 | CROPseq-RevExt-BgL-BsmBI | TGCAGCATGCGTCTCAAAAC | |
| 3.1 | 10X PrimeF-BgL-BbsI | GCCTGAAGACCTCACCGNNNNNNNNNNNNNNNNNNNNGTTTTAGTCTTCCATGCTGC | The N20 sequence is a random string of oligonucleotides |
| 3.1 | 10X-RevExt-BgL-BbsI | TGCAGCATGGAAGACTAAAAC | |
| 3.2, 3.7 | PrimeR_cropseq | CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTC | The N6 sequence is where the i7 Illumina index should be placed |
| 3.2, 3.7 | PrimeR_10x | CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTAGGACCGGCCTTAAAGC | The N6 sequence is where the i7 Illumina index should be placed |
| 3.2, 3.7 | PrimeF-PAS | equimolar mixture of UniF PAS primers | |
| 3.2, 3.7 | PrimeF-PASx0 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCTTTATATATCTTGTGGAAAGGACGAAAC | |
| 3.2, 3.7 | PrimeF-PASx1 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCGCTTTATATATCTTGTGGAAAGGACGAAAC | |
| 3.2, 3.7 | PrimeF-PASx2 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCGCTTTATATATCTTGTGGAAAGGACGAAAC | |
| 3.2, 3.7 | PrimeF-PASx3 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCGCTTTATATATCTTGTGGAAAGGACGAAAC | |
| 3.2, 3.7 | PrimeF-PASx4 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCAACGCTTTATATATCTTGTGGAAAGGACGAAAC | |
| 3.2, 3.7 | PrimeF-PASx5 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCACCGCTTTATATATCTTGTGGAAAGGACGAAAC | |
| 3.8 | BgN20-AB-fwd | TACTCGACCAAGAACCGCANNNNNNNNNNNNNNNNNNNNAGGTGGATTAGTTCTCT | Insert barcode in place of N20 |
| 3.8 | BgN20-AB-rev | AAGCAGAGAACTAATCCACCTNNNNNNNNNNNNNNNNNNNNTGCGGTTCTTGGTCG | Insert reverse-complement barcode in place of N20 |
| 3.8 | BgN20-BC-fwd | GCTTGTCCTGCGGTTACCCNNNNNNNNNNNNNNNNNNNNAGGCTGTAATCCAGCTG | Insert barcode in place of N20 |
| 3.8 | BgN20-BC-rev | AGCGCAGCTGGATTACAGCCTNNNNNNNNNNNNNNNNNNNNGGGTAACCGCAGGAC | Insert reverse-complement barcode in place of N20 |
| 3.8 | BgN20-CD-fwd | CGCTGTGGTATCACTCGTCNNNNNNNNNNNNNNNNNNNNAGGCTCAGCTAAGGTGC | Insert barcode in place of N20 |
| 3.8 | BgN20-CD-rev | CATTGCACCTTAGCTGAGCCTNNNNNNNNNNNNNNNNNNNNGACGAGTGATACCAC | Insert reverse-complement barcode in place of N20 |
Oligonucleotides
| Name | Vendor | Catalog No. |
|---|---|---|
| AMPure XP Reagent | Beckman Coulter | A63880 |
| BbsI-HF | NEB | R3539S |
| BsmBI-v2 | NEB | R0739S |
| Q5 Hot Start Polymerase | NEB | M0493S |
| T4 Ligase | NEB | M0202S |
| T4 PNK | NEB | M0201S |
| T7 Ligase | NEB | M0318S |
| Genomic DNA Mini Kit | ThermoFisher | K182001 |
| Lipofectamine 2000 | ThermoFisher | 11668030 |
| Lipofectamine 3000 | ThermoFisher | L3000001 |
| OptiMEM | ThermoFisher | 31985070 |
| Typan Blue Stain | ThermoFisher | T10282 |
| 2xYT medium | Millpore Sigma | Y2377-250G |
| Carbenicillin | Millpore Sigma | C1389-250MG |
| DMEM | Millpore Sigma | D5671 |
| Hexadimethrine bromide | Millpore Sigma | TR-1003-G |
| Plasmid Plus Kit (Midi) | Qiagen | 12941 |
| DNA Clean and Concentrator-5 | Zymo | D4013 |
Recommended Reagents