Appendix 1—key resources table.
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (H. sapiens) | UPF1 | Ensembl | ENSG00000005007 | |
| Gene (M. musculus) | Upf1 | Ensembl | ENSMUSG00000058301.8 | |
| Strain, strain background (M. musculus) | Mouse | NCI Charles River | Charles River: C57BL/6 albino | Mice for pancreatic injections |
| Strain, strain background (E. coli) | NEB Stable Competent E. coli (High efficiency) | New England Biolabs | C3040 | Chemically competent |
| Genetic reagent (M. musculus) | AdUpf1 | This paper | N/A | Adenovirus expressing CRISPR gRNAs targeting mouse Upf1 introns 9-11 |
| Cell line (H. sapiens) | HEK 293T | ATCC | CRL-11268 | Human cell line to model patient mutations |
| Cell line (M. musculus) | KPC | Generated from the PDX-1-Cre; LSL-KrasG12D/+; LSL Trp53R172H/+ | N/A | Murine cell line to model Upf1 exon skipping mutations (Hingorani et al., 2005) Provided by Robert Vonderheide |
| Antibody | Anti-human UPF1 (rabbit monoclonal) |
Abcam | Cat No. ab10936 | WB: (1:1000) |
| Antibody | Anti-human GAPDH (rabbit polyclonal) | Abcam | Cat No. ab9485 | WB: (1:1000) |
| Antibody | Anti-rabbit secondary antibody (goat monoclonal) | Abcam | Cat No. ab216777 | WB: (1:10000) IRDye 680RD |
| Antibody | Anti-mouse UPF1 (rabbit monoclonal) | Cell signaling technology | Cat No.9435 | WB: (1:1000) |
| Antibody | Anti-mouse tubulin (mouse monoclonal) | Sigma-Aldrich | Cat No. T9206 | WB: (1:2000) |
| Antibody | Anti-rabbit secondary antibody (from donkey) | Amersham | NA93V | WB: (1:5000) |
| Antibody | Anti-mouse secondary antibody (from sheep) | Amersham | NA931 | WB: (1:5000) |
| Antibody | Anti-mouse p40-ΔNp63 (rabbit polyclonal) | Abcam | Cat No. ab166857 | WB: (1:100) |
| Recombinant DNA reagent | pX459/Cas9 expression plasmid | Addgene | Cat No. 48139 | Ran et al., 2013 |
| Recombinant DNA reagent | phCMV-MUP | PMID:9671053 | Plasmid | Control for transfection efficiency of pmCMV-GI-Norm and pmCMV-GI-39Ter |
| Recombinant DNA reagent | pmCMV-Gl-Norm | PMID:9671053 | Plasmid | Transient transfection construct coding for full-length β-globin |
| Recombinant DNA reagent | pmCMV-Gl-39Ter | PMID:9671053 | Plasmid | Transient transfection construct coding for truncated β-globin with PTC at amino acid 39 |
| Sequence-based reagent | mUpf1_F | This paper | PCR primer | GGTGATGAGATTGCTATTGAGC |
| Sequence-based reagent | mUpf1_R | This paper | PCR primer | TGTTCCTGATCTGGTTGTGC |
| Sequence-based reagent | mUpf1-intron_9-gDNA_F | This paper | Guide DNA Oligo | CACCGTTGTGAGGGCCATACCCTTG |
| Sequence-based reagent | mUpf1-intron_9-gDNA_R | This paper | Guide DNA Oligo | AAACCAAGGGTATGGCCCTCACAAC |
| Sequence-based reagent | mUpf1-intron_11-gDNA_F | This paper | Guide DNA Oligo | CACCGCCGTTGAGCTGATGGTGGCT |
| Sequence-based reagent | mUpf1-intron_11-gDNA_R | This paper | Guide DNA Oligo | AAACAGCCACCATCAGCTCAACGGC |
| Sequence-based reagent | hUPF1_F | This paper | Genomic DNA PCR primer | AAAACGTTTGCCGTGGATGAG |
| Sequence-based reagent | hUPF1_R | This paper | Genomic DNA PCR primer | CACATAGAGAGCGGTAGGCA |
| Sequence-based reagent | hUPF1-gDNA_F | This paper | Guide DNA oligo | GCGCGCGGGGCCTCGCCCAT |
| Sequence-based reagent | hUPF1-patient_1-HDR_R | This paper | DNA HDR ultramer | GCTCAGTGGTCTTTGCAGCACAGTCTTCACGGCATAAACCTTCAATACAAGCGGCCGTTAGGGGCAGCCTCCGCTTGCGTCCCGGGCCATGGGTGAGGCCCCGCGCGCTGAGGACGGCGCGCACCTG |
| Sequence-based reagent | hUPF1-patient_9-HDR_R | This paper | DNA HDR ultramer | GCTCAGTGGTCTTTGCAGCACAGTCTTCACGGCATAAACCTTCAATACAAGCGGCTGTTAGGGGCAGCCTCCGCTTGCGTCCCGGGCCATGGGCGAGGCCCCGCGCGCTGAGGACGGCGCGCACCTG |
| Sequence-based reagent | hUPF1-PAM-control-HDR_R (wild type) | This paper | DNA HDR ultramer | GCTCAGTGGTCTTTGCAGCACAGTCTTCACGGCATAAACCTTCAATACAAGCGGCCGTTAGGGGCAGCCTCCGCTTGCGTCCCGGGCCATGGGCGAGGCCCCGCGCGCTGAGGACGGCGCGCACCTG |
| Sequence-based reagent | hUPF1-RT-PCR_F | This paper | RT-PCR primer | GGATGAGATATGCCTGCGGT |
| Sequence-based reagent | hUPF1-RT-PCR_R | This paper | RT-PCR primer | TTCTCGTCGGCAGACGACAG |
| Sequence-based reagent | Positive control gBlock to detect UPF1 splice variant (ΔE10-11) | This paper | DNA gBlock | ACATGCGGCTCATGCAGGGGGATGAGATATGCCTGCGGTACAAAGGGGACCTTGCGCCCCTGTGGAAAGGGATCGGCCACGTCATCAAGGTCCCTGATAATTATGGCGATGAGATCGCCATTGAGCTGCGGAGCAGCGTGGGTGCACCTGTGGAGGTGACTCACAACTTCCAGGTGGATTTTGTGTGGAAGTCGACCTCCTTTGACAGGCCGGTGCTGGTGTGTGCTCCGAGCAACATCGCCGTGGACCAGCTAACGGAGAAGATCCACCAGACGGGGCTAAAGGTCGTGCGCCTCTGCGCCAAGAGCCGTGAGGCCATCGACTCCCCGGTGTCTTTTCTGGCCCTGCACAACCAGATCAGGAACATGGACAGCATGCCTGAGCTGCAGAAGCTGCAGCAGCTGAAAGACGAGACTGGGGAGCTGTCGTCTGCCGACGAGAAGCGGTACCGGGCCTTGAAGCGCACCGCAGAGAGAGAGCTGCTGATG |
| Sequence-based reagent | mMup1-qPCR_F | PMID:25564732 | qPCR primer | GACCTATCCAATGCCAATCG (exon 5/6 junction) |
| Sequence-based reagent | mMup1-qPCR_R | PMID:25564732 | qPCR primer | GATGATGGTGGAGTCCTGGT (exon 7) |
| Sequence-based reagent | hβ-globin-qPCR_F | PMID:25564732 | qPCR primer | GCTCGGTGCCTTTAGTGATG (exon 2) |
| Sequence-based reagent | mβ-globin-qPCR_R | PMID:25564732 | qPCR primer | CCCAGCACAATCACGATCATA (exon 3, mouse specific) |
| Commercial assay or kit | RNeasy Plus Mini Kit | Qiagen | Cat No. 79654 | |
| Commercial assay or kit | Zero Blunt TOPO PCR cloning system | ThermoFisher | Cat No. K280020 | |
| Chemical compound, drug | Phosphatase inhibitor | ThermoFisher | Cat No. A32959 | |
| Chemical compound, drug | Protease inhibitor | Thermofisher | Cat No. A32963 | |
| Chemical compound, drug | Penicillin/Streptomycin | GIBCO | Cat No. 15070063 | |
| Chemical compound, drug | Lipofectamine 2000 | ThermoFisher | Cat No. 11668030 | |
| Chemical compound, drug | Lipofectamine 3000 | Invitrogen | Cat No. L3000001 | |
| Chemical compound, drug | Puromycin | ThermoFisher | Cat No. A1113803 | |
| Software, algorithm | GuideScan v1.0 | PMID:28263296 | http://www.guidescan.com/ | |
| Software, algorithm | Fiji v2.0.0 | ImageJ | https://imagej.net/Fiji | |
| Software, algorithm | RSEM v1.2.4 | PMID:21816040 | deweylab.github.io/RSEM/RRID:SCR_013027 | |
| Software, algorithm | Bowtie v1.0.0 | PMID:19261174 | github.com/BenLangmead/bowtie/; RRID:SCR_005476 | |
| Software, algorithm | TopHat v2.0.8b | PMID:19289445 |
ccb.jhu.edu/software/tophat/index.shtml
RRID:SCR_013035 |
|
| Software, algorithm | MISO v2.0 | PMID:21057496 | genes.mit.edu/burgelab/miso/ RRID:SCR_003124 | |
| Software, algorithm | IGV v2.3.90 | Thorvaldsdottir | software.broadinstitute.org/software/igv/ RRID:SCR_011793 | |
| Software, algorithm | Prism v7.0 | GraphPad Prism v7.0 |
http://www.graphpad.com/
RRID:SCR_002798 |
|
| Other | SuperScript IV Reverse Transcriptase | ThermoFisher | Cat No. 18090010 | |
| Other | Q5 High-Fidelity DNA Polymerase | New England Biolabs | Cat No. NEB #M0491 | |
| Other | PowerUp SYBER Green Master Mix | ThermoFisher | Cat No. A25742 | |
| Other | RNase-Free DNase Set | Qiagen | Cat No. 79254 |