title: SLX-14877.i718_i515
organism: Mus musculus
description: Protocols: To generate the iETO2-GLIS2:rtTA (iEG) model, the Tet Response Element (TRE) and the human EG cDNA was integrated at the Hprt locus on chromosome X . EG expression is controlled by a reverse tetracycline-controlled transactivator (rtTA) integrated into the Rosa26 gene locus and by doxycycline induction. The exact genotype is ETO2-GLIS2/y:rtTA/wt (male) or ETO2-GLIS2/ETO2-GLIS2:rtTA/wt (female). Transgenic mice ETO2-GLIS2/y:wt/wt or wt/y:rtTA/wt treated with doxycyclin but not expressing ETO2-GLIS2 have the phenotype no ETO2-GLIS2 expression. E14.5 fetal liver cells from CTRL and iETO2-GLIS2 (iEG) embryos were stained for flow cytometry using standard protocols and the LT-HSC populations were flow sorted according to the following markers : LT-HSC: LIN-Kit+Sca1+CD135-CD34-CD48-CD150+ MPP4: LIN-Kit+Sca1+CD135+CD34+CD48+CD150-. . Cell populations were flow sorted, cultured 24 hours in RPMI supplemented with 10% FBS, murine cytokines (IL3, IL6, SCF, TPO, FLT3L) and 100ng/ml of Doxycycline to induce ETO2-GLIS2 expression. 24 hours post-induction the 2 populations (CTRL LT-HSC, iEG LT-HSC) were individually sorted as single cell directly into the 2.4µl of lysis buffer in a 96-well plate using a FACS ARIA III Single cell were sorted directly into 2.4µl of lysis buffer in a 96-well plate.Reverse transcription (Superscript II RT) and amplification PCR (KAPA HiFi Hotstart ReadyMix) was performed in the same plate. PCR products were leaned using Warm Agencourt AMPure® XP beads and then supernatant was finally transferred into a fresh tube. Protocol was performed as previously described , check(Nestorowa et al. 2016 Blood) Tagmentation is carried out using the Illumina Nextera XT DNA sample preparation kit (Fluigdim). Then every 96 libraries were single pooled into a 1.5 ml eppendorf and cleaned using Warm Agencourt AMPure® XP beads. Quality of the pooled library was checked with an Agilent high-sensitivity DNA chip and quantified with KAPA library quantification kit. Protocol was performed as previously described (Nestorowa et al. 2016 Blood)
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-10-15T04:03:42Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-09-30T13:57:45Z
attributes.tag: External Id
attributes.value: SAMEA5983936
attributes.tag: INSDC center name
attributes.value: INSERM, Paris, France Equipe Labellisee Ligue Contre le Cancer Institut Gustave Roussy, Villejuif, France Universite Paris Diderot, Paris, France
attributes.tag: INSDC first public
attributes.value: 2019-10-15T04:03:42Z
attributes.tag: INSDC last update
attributes.value: 2019-09-30T13:57:45Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8360:SLX-14877.i718_i515
attributes.tag: age
attributes.value: embryonic day 14.5
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: hematopoietic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: facs marker
attributes.value: LIN-Kit+Sca1+CD135-CD34-CD48-CD150+
attributes.tag: genotype
attributes.value: ETO2-GLIS2/y:rtTA/wt; ETO2-GLIS2/ETO2-GLIS2:rtTA/wt
attributes.tag: organism part
attributes.value: liver
attributes.tag: phenotype
attributes.value: no ETO2-GLIS2 expression
attributes.tag: post analysis well quality
attributes.value: OK
attributes.tag: sample name
attributes.value: E-MTAB-8360:SLX-14877.i718_i515
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: not available
attributes.tag: strain
attributes.value: C57BL/6