Journal: iScience
Article Title: Excess of guide RNA reduces knockin efficiency and drastically increases on-target large deletions
doi: 10.1016/j.isci.2023.106399
Figure Lengend Snippet: Key resources table
Article Snippet: Cas9 Nuclease V3 Integrated DNA Technologies 1081058 Alt-R® CRISPR-Cas9 tracrRNA Integrated DNA Technologies 1072532 Alt-R® CRISPR-Cas9 crRNA, see Table S4 Integrated DNA Technologies custom DraIII-HF®restriction enzyme New England Biolabs R3510 Fixable Viability Stain 440UV BD Bioscience 566332 PMSG Med'Vet SYNCRO-PART PMSG 500 UI hCG Med'Vet CHORULON® 1500 UI XbaI restriction enzyme New England Biolabs R0145 Critical Commercial Assays P3 Primary Cell Nucleofector™ Solution Lonza V4XP-3032 Herculase II fusion Enzyme with dNTP Combo Agilent Technologies 600677 ddPCR supermix for probes (no dUTP) Bio-Rad 1863024 DNA 1K/12K/Hi Sensitivity Assay LabChip Perkin Elmer 760517 DNA High Sensitivity Reagent Kit Perkin Elmer CLS760672 Master Mix Fast SYBR™ Green Applied Biosystems 4385612 Experimental Models: Cell Lines LON71-019 (Gaignerie et al., 26 2018) N/A Experimental Models: Organisms/Strains Sprague-Dawley rats Janvier Labs RjHan:SD Oligonucleotides ssODN, see Table S4 Integrated DNA Technologies Ultramer® DNA Oligo, standard desalting HMA-CE and qPCR primers, see Table S4 Eurofins custom ddPCR-VIC labeled reference Sdha assay, see Table S4 ThermoFisher Scientific custom ddPCR FAM-labelled custom rat rEphx2 assays, see Table S4 Integrated DNA Technologies custom Software and Algorithms PR.Therm Control Software NanoTemper https://nanotempertech.com/prometheus-pr-thermcontrol-software/ FlowJo FlowJo software https://www.flowjo.com/ QuantaSoft Analysis Pro™ software Bio-Rad https://www.bio-rad.com/fr-fr/life-science/digital-pcr/qx200-droplet-digital-pcr-system/quantasoft-software-regulatory-edition GraphPad Prism 7.0 phPad Software, Inc http://www.graphpad.com/ Rstudio 2022.07.2 Posit https://posit.co/download/rstudio-desktop/ LabChip GX Perkin Elmer https://www.perkinelmer.com/fr/lab-products-and-services/resources/labchip-and-optimizer-software-downloads.html Other nanoDSF grade high sensitivity capillaries NanoTemper Technologies PR-C006 Matrigel® Matrix Corning 354277 mTeSR1 medium Stem Cell Technologies Catalog #85850 StemMACSTM Miltenyi Biotec 130-104-368 TrypLE™ Express Enzyme Thermofisher Scientific 12605010 Rock inhibitor Y 27632 dihydrochloride Axon Medchem 1683 StemFlex Thermofisher Scientific A3349401 M16 medium Sigma-Aldrich M7292 Droplet generation oil for probes Bio-Rad 1863005 Droplet generator DG8 Gasket Bio-Rad 1863009 DG8 Cartridges for QX100/QX200 droplet generator Bio-Rad 1864008 Automated droplet generation oil for probes Bio-Rad 1864110 DG32 cartridge for automated droplet generator Bio-Rad 1864108 Pierceable foil heat seal Bio-Rad 1814040 ddPCR droplet reader oil Bio-Rad 1863004 Twin-tec PCR plate 96, semi-skirted, red Eppendorf 30128613 Open in a separate window Key resources table .
Techniques: Recombinant, CRISPR, Staining, Sensitive Assay, SYBR Green Assay, Labeling, Software, Nano Differential Scanning Fluorimetry