2025-04-26
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA114190151
External Id
ED
INSDC center name
2023-07-24T10:25:55Z
INSDC last update
public
INSDC status
ERS16170847
SRA accession
E-MTAB-13169:Sample 11
Submitter Id
ArrayExpress
broker name
Cas9 TNG MKOS
cell_line
mouse embryonic fibroblast
cell_type
not collected
collection date
house mouse
common name
Protocols: For control and Hic2 overexpressing MEF samples, 1 × 105 Cas9 TNG MKOS MEFs were transduced with either Ty1-BFP (control, BFP tagged with a Ty1 tag at the N-term end ) or Ty1-Hic2 (HIC2 tagged with a Ty1 tag at the N-term end) lentiviruses at an MOI of 3 with 8 µg/ml polybrene (Merck-Millipore) for 4 h. After 3 days in culture in MEF media, the cells were harvested and FACS-sorted to exclude dead cells with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (ThermoFisher Scientific, Dilution: 1/1500). For reprogramming samples, 0.5 × 104 Cas9 TNG MKOS MEFs were mixed with 9.5 × 104 WT MEFs (129 strain) and seeded in gelatin-coated wells of 6-well plates. Cells were transduced with either Ty1-BFP or Ty1-Hic2 lentivirus at an MOI of 3 with 8 µg/ml polybrene (Merck-Millipore) for 4h, before being recovered for 24 h in MEF media. Reprogramming was initiated by the addition of a reprogramming medium. Cells were harvested at day 2, day 4, day 6 and day 10 of reprogramming, respectively, and a maximum of 1 × 105 of mOrange+ OSKM and Nanog-GFP+ expressing cells were sorted with the FACS AriaII (BD Biosciences) per sample. Additionally, iPSCs were generated from Cas9 TNG MKOS MEFs transduced with a tet-inducible version of the Ty1-BFP and Ty1-Hic2 lentiviral particles Nanog-GFP+ iPSCs were harvested at day 15, and sorted with the FACS AriaII (BD Biosciences) and put in culture in ESC medium for 20 days in absence of doxycycline. All cells were homogenized with the QIAshredder kit (Qiagen) and total RNA was extracted from all samples using the RNeasy Plus Micro Kit (Qiagen). Libraries were prepped with the NEB Ultra II stranded mRNA Library prep kit (NEB).
description
mouse E12.5
developmental stage
normal
disease
BFP overexpression
genetic modification
Rosa-EF1a-Cas9, Nanog-GFP, Rosa-rtTA, Sp3-tetO-MKOS-ires-mOrange
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
embryo
organism part
Mus musculus
scientific_name
+doxycycline
stimulus
129 mouse strain
strain
Sample 11
title