Illumina MiSeq paired end sequencing; NGS data from 'Treatment of a metabolic liver disease by in vivo genome base editing in adult mice'
NGS data from 'Treatment of a metabolic liver disease by in vivo genome base editing in adult mice'
Transfected cells and organoids were collected following trypsinization and washed with 1x PBS, mouse livers were collected after euthansia using CO2 and washed with 1x PBS.   HEK293T cells were obtained from ATCC and verified mycoplasma free (GATC Biotech). The HEK293T reporter line was maintained in DMEM with GlutaMax (Gibco). Media was supplemented with 10% FBS (Sigma), and 100 μg/mL Penicillin-Streptomycin (Gibco). Mouse liver organoid cultures: Mouse liver tissue was mechanically dissociated and digested in Dulbecco's Modified Eagle's medium supplemented with Collagenase type IX 0.012%, Dispase 0.012%, and 1% (v/v) FBS for 30 min. Biliary ducts were identified in the microscope and seeded in 20µl Matrigel (Corning) drops. After gelation at 37°C, culture medium was added:  Advanced Dulbecco's Modified Eagle's medium supplemented with 10% (v/v) RSPO1-conditioned medium, 1 × B27 minus Vitamin A (Thermo Fisher Scientific), 1 × N-2 (Thermo Fisher Scientific), 10 mM Nicotinamine (Sigma-Aldrich), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 10 nM gastrin (Tocris Bioscience), 10 nM Gastrin (Tocris Bioscience), 50 ng/ml EGF (Preprotech), 50 ng/ml HGF (Preprotech) and 50 ng/ml FGF (Preprotech). During the first 4-5 days after isolation, organoid culture medium was supplemented with 10% (v/v) Noggin- and 50% (v/v) Wnt3a-conditioned medium. Organoids were passaged by mechanical dissociation and transferred into fresh Matrigel drops. The organoids were cultured in Expansion medium: Advanced Dulbecco's Modified Eagle's medium supplemented with 10% (v/v) RSPO1-conditioned medium prepared according to Farin et al.39, 1 × B27 minus Vitamin A (Thermo Fisher Scientific), 1 × N-2 (Thermo Fisher Scientific), 10 mM Nicotinamine (Sigma-Aldrich), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 10 nM gastrin (Tocris Bioscience), 10 nM Gastrin (Tocris Bioscience), 50 ng/ml EGF (Preprotech), 50 ng/ml HGF (Preprotech) and 50 ng/ml FGF (Preprotech). During the first 4-5 days after isolation, organoid culture medium was supplemented with 10% (v/v) Noggin- and 50% (v/v) Wnt3a-conditioned medium prepared according to Barker et al. (2010). Organoids were passaged by mechanical dissociation and transferred into fresh Matrigel drops. HEK293T cells were seeded in 48-well plates to reach approximately 70% confluency 12-16h after plating. 1 ug total plasmid DNA was transfected. Cells were incubated for 5 days and split into a 24-well cell culture plate (Greiner) 24 h post transfection with a media change on day 4.  Mouse liver organoids were dissociated into single cells using TrypLE (Thermo Fisher Scientific) at 37°C. After 5-6 min, three volumes of Dulbecco's Modified Eagle's Medium supplemented with GlutaMax (Thermo Fisher Scientific) and 10% (v/v) fetal bovine serum (FBS) were added, and samples were centrifuged. Cells were resuspended in supernatant containing lentiviral particles and Advanced Dulbecco's Modified Eagle's medium (Thermo Fisher Scientific) was added to a total of 500 µl (including lentiviral particles) and plated in 24 well suspension plates (Greiner). The plate was centrifuged for one hour at 32°C and 700 g, followed by a three-hour incubation at 37°C and 5% CO2. Single cells were collected replated in 20 µl Matrigel (Corning) drops. Culture medium supplemented with 10μM Y-27632 (Tocris Bioscience) was added. After 2 days, stably transduced organoids were enriched with 3 µg/ml Puromycin (Invivogen) for 7 days. Supernatant containing lentiviral particles was added to HEK293T and Hepa1-6 cells in a 24 well cell culture plate (Greiner). Two days after transduction, cells were enriched using 2.5µg/ml Puromycin for seven days before sequencing analysis.  Mice were administered two AAVs from 5E+10 to 1E+12 per AAV 4 weeks or more before sequencing analysis.    DNA extraction was done using DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's protocol. RNA was isolated using the RNeasy mini kit (Qiagen) following the manufacturer's protocol. RNA was reverse transcribed using the GoScript reverse transcriptase (Promega) according to the manufacturer's protocol  PCR amplicons flanking the targeted site were generated using NEBNext High-Fidelity 2X PCR Master Mix (NEB). PCR cycling conditions were as follows: 1x 98°C for 3 min; 26x 98°C for 15s, 65°C for 15s, 72°C for 8s; 1x 72°C for 2min for 200ng gDNA. The same cycling protocol but with 22 cycles was used for amplification from cDNA (from 50ng RNA). PCR amplicons were purified by solid phase reversible immobilization (SPRI) bead cleanup using Agencourt AMPure XP reagent (A63881, Beckmann-Coulter, Indianapolis, IN, USA), according to the manufacturer's instructions. For generation of pooled sequencing libraries, the TruSeq (Illumina) Index Adaptor Sequences were added in a second amplification step. The resulting Illumina libraries with Index Adaptors were gel extracted (Nucleospin, Machery Nagel) according to the manufacturer's instructions. Quality control for the final library was performed using the High sensitivity D1000 ScreenTape at Agilent 2200 TapeStation.
Experimental Factor: targeting method
in vivo
Experimental Factor: material for PCR amplification
genomic DNA
Experimental Factor: PCR amplicon
off target
Experimental Factor: base editing
performed
Experimental Factor: base editor
full length
Experimental Factor: guide RNA
sgRNA targeting Pah gene