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Contents:In the table below, Illumina Nextera Handle sequences have been added and highlighted in bold. Primers for the on-target have been added for convenience. The table below is sorted by the CFD off-target score. Sites with very low CFD scores < 0.02 are unlikely to be cleaved, see our study Haeussler et al. 2016, Figure 2.
In the protocol by Matthew Canver, Harvard, two PCRs are run: one PCR to amplify the potential off-target, then a second PCR to extend the handles with Illumina barcodes. Please click here to download the protocol. Alternatively, you can have a look at Fu et al, 2014.
If a primer was not found, the reason is usually that the region around the off-target is too repetitive. To avoid unspecific primers, all repeats are masked for the primer design (not for off-target search). If you think that we should change the parameters here or should use different primer3 settings, please let us know.
| Name | Primer Sequence | Tm | CFD Score |
|---|---|---|---|
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313535_F | TCGTCGGCAGCGTCCATTAGCTGCGGGTCTCCTT | 59.8 | 1.00 |
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313535_R | GTCTCGTGGGCTCGGCTGTCATCCTCCTGCTGCAA | 60.0 | 1.00 |
| mm4_intergenic_1700018A04Rik|Gm11378_chr13_31618695_F | TCGTCGGCAGCGTCTGCCATTTCCAAGAGCCTGG | 60.6 | 0.28 |
| mm4_intergenic_1700018A04Rik|Gm11378_chr13_31618695_R | GTCTCGTGGGCTCGGGCTTTTGTGTCTGCTCTGGC | 60.0 | 0.28 |
| mm4_intron_Galnt2_chr8_124280516_F | TCGTCGGCAGCGTCGAGATGGTTTGCATGCCAGT | 58.8 | 0.19 |
| mm4_intron_Galnt2_chr8_124280516_R | GTCTCGTGGGCTCGGGTTCAGGAATCGACTGCCCA | 60.0 | 0.19 |
| mm4_intergenic_Rpl31-ps15|Gm26404_chr3_37741626_F | TCGTCGGCAGCGTCTCCATTCAAGGGCAGGTCAC | 59.9 | 0.11 |
| mm4_intergenic_Rpl31-ps15|Gm26404_chr3_37741626_R | GTCTCGTGGGCTCGGTGCCTGCATGAGACATCAGG | 60.1 | 0.11 |
| mm4_intergenic_Irx5|Irx6_chr8_92465255_F | TCGTCGGCAGCGTCGAGAGAGGCCACCCAGATTC | 59.5 | 0.00 |
| mm4_intergenic_Irx5|Irx6_chr8_92465255_R | GTCTCGTGGGCTCGGGCTGAGAGAGAGGCTATGGC | 59.6 | 0.00 |
These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.
| ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313535 |
CATTAGCTGCGGGTCTCCTTTCATCTTCACTGTGGCAGACGTTTCTATTTATCCACTTGCGTTCGCCGAGTGGCGTCACC AGCGGTACTGTAATGACGATTGCAGCAGGAGGATGACAG |
| mm4_intergenic_1700018A04Rik|Gm11378_chr13_31618695 |
TGCCATTTCCAAGAGCCTGGCGCCACCCAGCGGACACAAGAGGAACACTGGGCGCGTCTCAGTTCCCCCAAGTCGCCAGA GCAGACACAAAAGC |
| mm4_intron_Galnt2_chr8_124280516 |
GAGATGGTTTGCATGCCAGTATAGTTGGACCCTCTTGCGTTTGCTGAGGGGAGGTGGTTGAGACGTTGTAACTGGGAAAA GCGTATATGATGGGCAGTCGATTCCTGAAC |
| mm4_intergenic_Rpl31-ps15|Gm26404_chr3_37741626 |
TCCATTCAAGGGCAGGTCACAACCTTCCAATGCTGAACAGTGTTCAAGAGAGAAATGTTCAGGAATTCCCTCAGATGAAG CATACATGACCCCCTTGTGGTCGCAGAGTGGCTGGGCTTCCCTGATGTCTCATGCAGGCA |
| mm4_intergenic_Irx5|Irx6_chr8_92465255 |
GAGAGAGGCCACCCAGATTCCCCCTTGGGTACACCGAGTGGCTGGAGTATCTATTATAGATGCCTCCCATCCCAATAAAT ATTTCAATGCAAATAAATAAAGGATGCCATAGCCTCTCTCTCAGC |