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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.
Name | Primer Sequence | Tm | CFD Score |
---|---|---|---|
ontarget_mm0__chr7 5.53 Mbp_chr7_5529716_F | TCGTCGGCAGCGTCTGGTGAGCTGCGAGAATAGC | 60.1 | 1.00 |
ontarget_mm0__chr7 5.53 Mbp_chr7_5529716_R | GTCTCGTGGGCTCGGCGGCTTCCTTTGTCCCCAAT | 60.6 | 1.00 |
mm4__chr14 70.42 Mbp_chr14_70417095_F | TCGTCGGCAGCGTCCACAGCGGTTTCGGTTTCAG | 60.0 | 0.49 |
mm4__chr14 70.42 Mbp_chr14_70417095_R | GTCTCGTGGGCTCGGAAACTGCAATCCATTGGCGG | 59.7 | 0.49 |
mm4__chr2 119.49 Mbp_chr2_119488100_F | Primer3: not found at this Tm | N.d. | 0.38 |
mm4__chr2 119.49 Mbp_chr2_119488100_R | Primer3: not found at this Tm | N.d. | 0.38 |
mm4__chr1 207.67 Mbp_chr1_207670930_F | TCGTCGGCAGCGTCACCTAGAAATTTTGTTGCTGGAGT | 58.8 | 0.21 |
mm4__chr1 207.67 Mbp_chr1_207670930_R | GTCTCGTGGGCTCGGTGCATACACCTTGGGAGCAT | 59.3 | 0.21 |
mm4__chr22 49.20 Mbp_chr22_49195866_F | TCGTCGGCAGCGTCGGCAACCTCCCTTGTCTGG | 60.3 | 0.19 |
mm4__chr22 49.20 Mbp_chr22_49195866_R | GTCTCGTGGGCTCGGTCAGTCTCGGGAGGAGTGAG | 60.0 | 0.19 |
mm4__chr17 78.59 Mbp_chr17_78590923_F | Primer3: not found at this Tm | N.d. | 0.10 |
mm4__chr17 78.59 Mbp_chr17_78590923_R | Primer3: not found at this Tm | N.d. | 0.10 |
mm4__chr18 80.08 Mbp_chr18_80084090_F | TCGTCGGCAGCGTCGGAACCGTGGAAGTGCTGAT | 60.3 | 0.10 |
mm4__chr18 80.08 Mbp_chr18_80084090_R | GTCTCGTGGGCTCGGGTTCCACTGACCTAGCAGGC | 60.3 | 0.10 |
mm4__chr10 117.55 Mbp_chr10_117553513_F | TCGTCGGCAGCGTCTTGAGAACCCTCTGACCCCA | 60.1 | 0.06 |
mm4__chr10 117.55 Mbp_chr10_117553513_R | GTCTCGTGGGCTCGGTGATGAATGGACCTTGCGCT | 60.0 | 0.06 |
mm4__chr3 143.97 Mbp_chr3_143972920_F | TCGTCGGCAGCGTCCTACGCGGAGACCAAGGAC | 59.8 | 0.04 |
mm4__chr3 143.97 Mbp_chr3_143972920_R | GTCTCGTGGGCTCGGCGCCTACCTGTAGCACCAG | 59.8 | 0.04 |
mm4__chr2 71.28 Mbp_chr2_71276505_F | TCGTCGGCAGCGTCCTGCAGGGGAGGAGCTGG | 61.7 | 0.03 |
mm4__chr2 71.28 Mbp_chr2_71276505_R | GTCTCGTGGGCTCGGCCTCAAGGCGCAGGACTC | 60.1 | 0.03 |
mm4__chr11 909.45 Kbp_chr11_909450_F | TCGTCGGCAGCGTCATAAGGTGGAGCGCTACTGC | 59.8 | 0.02 |
mm4__chr11 909.45 Kbp_chr11_909450_R | GTCTCGTGGGCTCGGTTCCATCAACACCACCGAGG | 59.9 | 0.02 |
mm4__chrY 276.92 Kbp_chrY_276917_F | TCGTCGGCAGCGTCCGTTGATTCACCCAGCAACC | 59.7 | 0.02 |
mm4__chrY 276.92 Kbp_chrY_276917_R | GTCTCGTGGGCTCGGCCAGGCGGGGTGAGACAC | 62.3 | 0.02 |
mm4__chr19 19.54 Mbp_chr19_19544633_F | TCGTCGGCAGCGTCGGCTTGTGGGAGGAGGAGA | 60.6 | 0.01 |
mm4__chr19 19.54 Mbp_chr19_19544633_R | GTCTCGTGGGCTCGGAGGCACGTCCAGATTGAACA | 59.6 | 0.01 |
mm4__chr14 96.36 Mbp_chr14_96363777_F | TCGTCGGCAGCGTCCGTGTTAAGAGGGGTCTGGG | 59.7 | 0.01 |
mm4__chr14 96.36 Mbp_chr14_96363777_R | GTCTCGTGGGCTCGGCTCGAGAAGGGCTCCAGTCC | 61.6 | 0.01 |
mm4__chr18 75.20 Mbp_chr18_75204856_F | TCGTCGGCAGCGTCGGGTGGACAAGTTCTCTCCG | 60.0 | 0.00 |
mm4__chr18 75.20 Mbp_chr18_75204856_R | GTCTCGTGGGCTCGGCCAAGGTCACGGTCAAGCC | 60.9 | 0.00 |
Primers underlined, off-targets in bold.
ontarget_mm0__chr7 5.53 Mbp_chr7_5529716 |
TGGTGAGCTGCGAGAATAGCCGGGCGCGCTGTGAGCCGAGGTCGCCCCCGCCCTGGCCACTTCCGGCGCGCCGAGTCCTT AGGCCGCCAGGGGGCGCCGGCGCGCGCCCAGATTGGGGACAAAGGAAGCCG |
mm4__chr14 70.42 Mbp_chr14_70417095 |
CACAGCGGTTTCGGTTTCAGCAGCCTCGAGACCCGGAAAAGGAAGCCCGAAGGACTCGGAGCACTGGGAATGCTGGGAAT CGTGGTTCCTACCGCCAATGGATTGCAGTTT |
mm4__chr1 207.67 Mbp_chr1_207670930 |
ACCTAGAAATTTTGTTGCTGGAGTAATTTCAATTTTAAAATGCTTATGGATGCTTCCGGTGTGCACAGTCCTTTGGAATT TAAAAGAAATGTAAGAGAAAGAGCTATGCTCCCAAGGTGTATGCA |
mm4__chr22 49.20 Mbp_chr22_49195866 |
GGCAACCTCCCTTGTCTGGGGGTGGCTATGGGGCTTTGGCTGCGCCGAGTCCTTTGGTGAGTGCTGCCCCGCTCACTCCT CCCGAGACTGA |
mm4__chr18 80.08 Mbp_chr18_80084090 |
GGAACCGTGGAAGTGCTGATGGGGAAATGACTCAATGTCCTTTACCTGAAAATCCCTAGGCAGGGATTTCCTGCGTGCAG AGTCCTAAGGATGCCTGCTAGGTCAGTGGAAC |
mm4__chr10 117.55 Mbp_chr10_117553513 |
TTGAGAACCCTCTGACCCCAGCCGTGACCCCTCTCCGGGAGGTGGCTTCTCCCGCAGGACCCGGTGCGCCAGAAAACTCT AGGCGGCGCCACGCTTGGAAGCGCAAGGTCCATTCATCA |
mm4__chr3 143.97 Mbp_chr3_143972920 |
CTACGCGGAGACCAAGGACTCGGGCAGCTTCCTGCTTCGCAACCTCAAGGACTCGGAGCGCATGCAGCTGCTGCTGACCC TGGCCTTCAACCCCGAGCCGCTGGTGCTACAGGTAGGCG |
mm4__chr2 71.28 Mbp_chr2_71276505 |
CTGCAGGGGAGGAGCTGGGCCCCGCCCCCGGCTTCCCGCGCTCCGCGTCCATTGGCTGGCCTCGGGGGTGGGCGGGTCCC GCCGCACGGAGTCCTGCGCCTTGAGG |
mm4__chr11 909.45 Kbp_chr11_909450 |
ATAAGGTGGAGCGCTACTGCTCCCACTTTCCTGCGCTCCCAGTCCGTAGGTCCAGGACCTCCTCGGTGGTGTTGATGGAA |
mm4__chrY 276.92 Kbp_chrY_276917 |
CGTTGATTCACCCAGCAACCATTCACCCCGTGCGTTGAATGTGCGCGTCACGGCCACTCACGCCTCTGGGACGTGAGTCT CCCGAGGACTCAGCGGGCGGGAAGGTGTAGGTGTCTCACCCCGCCTGG |
mm4__chr19 19.54 Mbp_chr19_19544633 |
GGCTTGTGGGAGGAGGAGAGCGGCTTCCGGCGCGAGGGGTCCTCGGGCCCCCGGGTGCGCCGGGAGGAGCGCGTCTTCCT GGTGGGCAACGTGGAGATCCGGGAGCGGCGCCTGTTCAATCTGGACGTGCCT |
mm4__chr14 96.36 Mbp_chr14_96363777 |
CGTGTTAAGAGGGGTCTGGGGGCCGGTCTGGCCGGCCTGGCTCTCCCGGCTCCCCTCAGACCTCGGCGCCCCGGAAACGC ACACTTGGGGCTCCGCCGTCTCCGCTGGGAGGACTGGAGCCCTTCTCGAG |
mm4__chr18 75.20 Mbp_chr18_75204856 |
GGGTGGACAAGTTCTCTCCGAGTCCCGTGCACTCGGCGCGCCGGAGCGCAGTGGCCGACAGACAGGGCCCGCCGGCGGCT TGACCGTGACCTTGG |