2024-07-19
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA112849753
External Id
EMBL
INSDC center name
2023-03-29T16:42:39Z
INSDC last update
public
INSDC status
ERS14856374
SRA accession
E-MTAB-12866:mRNA_K9A_A4
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. RNA was extracted from ~10 million cells using the RNeasy Mini kit (Qiagen) and DNase digestion was performed using TURBO DNase (Thermo Fisher Scientific), according to the manufacturer’s instructions and an additional clean-up of the RNA was then performed using the RNeasy Mini kit (Qiagen). Quality of the RNA was assessed on Bioanalyzer using the Agilent RNA 6000 Nano Kit and samples with a RNA integrity number (RIN) of 10 were used for library generation. For each sample, 100 ng of total RNA was used as input for mRNA selection and conversion to cDNA using the NEBNext Poly(A) Magnetic Isolation Module (New England BioLabs). Sequencing libraries were prepared using the NEBNext Ultra II RNA Library Preparation Kit for Illumina (New England BioLabs), according to the manufacturer’s instructions. After Qubit quantification, the quality of individual libraries was assessed on a Bioanalyzer using the Agilent High Sensitivity DNA Kit.
description
targeted mutation
genetic modification
H3f3a knockout; H3f3b K9A
genotype
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
Mus musculus
scientific_name
frozen specimen
specimen with known storage state
mRNA_K9A_A4
title