III_ESC_mR26-2
Mus musculus
Protocols: Transfected cells were collected by trypsinization and were washed with 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). K562 cells were obtained from Sigma Aldrich, verified mycoplasma free and were maintained in RPMI 1640 medium with GlutaMax. Additional the medium supplemented with 10% FBS, and 100 μg/mL Penicillin-Streptomycin. Cells were passaged three times per week. Cells were grown at 37C in a humidified 5% CO2 environment. WT E14 mESC line (ATCC CRL-1821) was cultured in Dulbecco's Modified Eagle Media (DMEM) (Sigma-Aldrich), containing 15% of fetal bovine serum (FBS; Life Technologies), 100 U/mL LIF (Millipore), 0.1 mM 2-ß-mercaptoethanol (Life Technologies) and 1% Penicillin/Streptomycin (Gibco), on 0.2% gelatin-coated support in absence of feeder cells. The culture medium was changed daily. HEK293T cells were seeded in 24-well plates at 120.000 - 140.000 cells per well, 1 day prior to transfection. K562 cells were 6h prior to transfection distributed in 24 well plates at a density of 220.000- 240.000 cells per well. On the day of transfection, RNP and RNPD complexes (2.2 pmols) were complexed with sgRNA (3.88 pmols) in Opti-MEM (Invitrogen) and briefly vortexed, followed by adding 3 μl the Lipofectamine 2000 reagent (Invitrogen) with Opti-MEM. The resulting mixture was incubated for 15 min at room temperature to allow lipid particle formation. After 15 minutes of incubation at room temperature, the mixture was dropped slowly into the well. One day post-transfection, cells were transferred to an 10cm dish. mESCs were transfected into 6-well plate using Lipofectamine 2000. Cells were plated 24 hours before transfection at a density of 20,000 cells/cm2 per well and cultured in culture medium without streptomycin and penicillin. The medium was changed to mESC culture medium 8 hours after transfection. Cells were collected 48h post-transfection. The expression of SpCas9-SNAP protein necessary for RNP/RNPD complexes was done from bacterial expression vector pNS20-SpCas9-SNAP. Wild Type Streptococcus pyogenes Cas9 (SpCas9 WT) proteins were expressed in Escherichia coli BL21 (DE3) Rosetta 2 from plasmid pMJ806. Bacterial expression plasmid pMJ806 was a gift from Jennifer Doudna (Addgene plasmid # 39312). In the RNP sample the system contains SpCas9-SNAP protein complexed with the sgRNA, but no repair oligo. PBS was discarded and DNA extraction was preformed using DNeasy Blood and Tissue kit (Qiagen) following manufacturer's protocol. The PCR amplicons flanking the targeted site were generated using NEBNext® High-Fidelity 2X PCR Master Mix (NEB). PCR cycling conditions used were as follows: 1x 98°C for 3 min; 27x 95°C for 15 s, 65°C for 15 s, 72°C for 30 s; 1x 72°C for 5 min. Annealing temperature was optimized for each primer set to ensure that a single amplicon was produced. PCR amplicons were purified by solid phase reversible immobilization (SPRI) bead cleanup using Agencourt AMPure XP reagent (#A63881, Beckmann-Coulter, Indianapolis, IN, USA), per the manufacturer's instructions. For the generation of the pooled sequencing libraries, the TruSeq (Illumina®) Index Adaptor Sequences were added at the second amplification step. The resulting Illumina libraries with Index Adaptors were purified with AMPure XP reagent. Quality control for the final library was performed using the High sensitivity D1000 ScreenTape at Agilent 2200 TapeStation.
ENA-FIRST-PUBLIC
2018-06-04T17:01:56Z
ENA-LAST-UPDATE
2018-05-25T09:13:19Z
External Id
SAMEA4688254
INSDC center name
Statistical Genomics group Institute of Molecular Life Sciences
INSDC first public
2018-06-04T17:01:56Z
INSDC last update
2018-05-25T09:13:19Z
INSDC status
public
Submitter Id
E-MTAB-6808:III_ESC_mR26-2
amplicon
mRosa26
broker name
ArrayExpress
cell line
ES cell line
common name
house mouse
experimental condition
RNP
genotype
wild type genotype
replicate
3
sample name
E-MTAB-6808:III_ESC_mR26-2
scientific_name
Mus musculus