OHT_rep1
Mus musculus
Protocols: Total RNA was purified by RNeasy Minikit (Qiagen) according to the manufacturer's manual. RNA concentration was determined by Nanodrop, and 500 ng of total RNA was used for library construction using KAPA Stranded mRNA-Seq Kits. Sequencing was performed on Illumina HiSeq 2500 machines with 125 bp pair-end mode. The Dpf2 targeting vector was linearized and electroporated into R26::CreERT2 E14 cells to generate heterozygous ES cell lines after G418 selection. The selected heterozygous ES lines were transiently transfected with a FLP recombinase encoding plasmid (pCAGGsFlpE), converting the initial knockout allele (Dpf2+/-, lacZ positive, G418 resistant) into a “wild-type” (WT) allele with two floxP sites flanking exon 4 (Dpf2+/+, reverted WT (rWT), lacZ negative, G418 sensitive). Multiple independent rWT ES clones were then electroporated with the original Dpf2 knockout vector or Dpf2 critical exon-deleted targeting vector and selected with G418. Dpf2 critical-exon-deleted vector was generated by transforming Dpf2 targeting vector into the cre-containing bacteria. In the second round targeting, the clones recovered were only those with the second WT allele being targeted, which were confirmed by the presence of both the rWT allele and the second knockout allele. The long-range PCR reactions were carried out for genotyping (SequalPrep Long PCR kit, Invitrogen). The selected heterogeneous ES cell lines were converted to the conditional Dpf2+/+ and Dpf2+/- clones by transiently transfected with FlpE. RNeasy Minikit (Qiagen) KAPA Stranded mRNA-Seq Kits
ENA first public
2018-06-30
ENA last update
2017-10-31
External Id
SAMEA104364244
INSDC center alias
Wellcome Trust Sanger Institute
INSDC center name
Wellcome Trust Sanger Institute
INSDC first public
2018-06-30T17:01:47Z
INSDC last update
2017-10-31T17:01:13Z
INSDC status
public
Submitter Id
E-MTAB-6166:OHT_rep1
broker name
ArrayExpress
cell line
E14-ES
cell type
embryonic stem cell
common name
house mouse
disease
normal
genotype
Dpf2 flox/flox
organism part
inner cell mass
sample name
E-MTAB-6166:OHT_rep1