2024-09-05
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA112849771
External Id
EMBL
INSDC center name
2023-03-29T17:05:11Z
INSDC last update
public
INSDC status
ERS14856392
SRA accession
E-MTAB-12867:DNAseq_K79A_cl5
Submitter Id
ArrayExpress
broker name
ESC 129-B13
cell_line
embryonic stem cell
cell_type
house mouse
common name
Protocols: Mouse embryonic stem cells (129XC57BL/6J) were cultured in ESC medium containing Knockout-DMEM (Thermo Fisher Scientific) supplemented with 15% EmbryoMax fetal bovine serum (ES-FBS Merck-Millipore) and 20 ng/ml leukemia inhibitory factor (LIF, produced by Protein Expression and Purification Facility at EMBL Heidelberg), 1% non-essential amino acids (Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 1% Pen/Strep (Thermo Fisher Scientific), 1% of 55 mM beta-Mercaptoethanol solution (Merck Millipore). Upon thawing, cells were cultured on a layer of mouse embryonic fibroblasts (MEFs) and afterwards passaged on 0.1% gelatine-coated culture dishes, without feeder cells. Cells were maintained at 37°C with 5% CO2 and routinely tested for mycoplasma. The medium was changed daily, and cells were passed every two days. Genomic DNA was extracted from ~10 million cells using the Gentra Puregene Cell Kit (Qiagen). Genomic DNA was extracted from ~10 million cells using the Gentra Puregene Cell Kit (Qiagen) and sonicated at 4°C using a Bioruptor Pico sonication device (Diagenode) for 10 cycles (30s ON/30s OFF) to obtain fragments of ~200 bp. The fragmentation pattern was assessed via agarose gel electrophoresis and 0.5-1 μg of fragmented gDNA was used to prepare sequencing libraries using the NEBNext Ultra II DNA Library Preparation Kit (New England Biolabs), according to manufacturer’s instructions. Libraries were then quantified using the Qubit fluorometer (Thermo Fisher Scientific) and the quality was assessed on a Bioanalyzer using the Agilent High Sensitivity DNA Kit.
description
targeted mutation
genetic modification
H3f3a knockout; H3f3b K79A
genotype
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
Mus musculus
scientific_name
fresh specimen
specimen with known storage state
DNAseq_K79A_cl5
title