characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2014-05-15T17:02:21Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T17:34:16Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2496545
characteristics.characteristic: External Id
characteristics.text: European Molecular Biology Laboratory
characteristics.characteristic: INSDC center name
characteristics.text: 2014-05-15T17:02:21Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T17:34:16Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS449473
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2555:E3.5_ICM_2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Embryos were collected at the early blastocyst stage (E3.5) by uterine flush in M2 medium (Sigma). Embryos were subjected to immunosurgery (Solter and Knowles, Proc Natl Acad Sci USA 72:5099-102; Nichols et al. Cell 95:379-91). Briefly, blastocysts were incubated for 15min in a 1:5 dilution of anti-mouse rabbit serum (Sigma) in N2B27 medium, washed in N2B27 and further incubated for 15min in a 1:5 dilution of rat serum (in-house) in N2B27 for the complement reaction. Embryos were transferred into N2B27 without washing and incubated for 15min to allow efficient cell lysis. The ICM was subsequently cleaned from residual trophectoderm with a narrowly fitting glass pipette. ICM cells were transferred immediately into 4.5ul of single-cell lysis buffer under a dissection scope and snap frozen on dry ice. cDNA synthesis was performed using whole transcriptome preamplification (Tang et al. Nat Methods 6:377-82; Tang et al. Cell Stem Cell 6:468-78). PCR was carried out for 20 cycles using a single-temperature protocol in a Veriti 96 Well Thermal Cycler (Applied Biosystems). Preamplified cDNA samples were sheared by ultrasonication on a Covaris S2 for 80s with the following parameter settings: Duty Cycle = 10, Cycles per Burst = 200, Intensity = 4. Samples were ethanol precipitated and resuspended in Tris buffer for subsequent enzymatic modifications. End repair was carried out with T4 DNA polymerase and T4 polynucleotide kinase (New England Biolabs) and samples were purified with Ampure XP paramagnetic beads (Beckman Coulter). Blunt-end, 3'-phosphorylated products were 3'-adenylated with exo- Klenow fragment in the presence of dATPs (New England Biolabs), purified with Ampure beads, and ligated to sequencing adapters (Illumina) by T4 DNA ligase at 20C for 30 minutes. PCR amplification of library constructs was carried out with AccuPrime DNA polymerase (Invitrogen) for 13 cycles on a Bio-Rad C1000 thermocycler using denaturing and annealing conditions optimised for even A/T vs. G/C processing (Aird et al. Genome Biol 12:R18). Molarity and size distribution of sequencing libraries were assessed by DNA 1000 microfluidic chips on the 2100 Bioanalyzer (Agilent).
characteristics.characteristic: description
characteristics.text: embryonic day 3.5
characteristics.characteristic: developmental stage
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: inner cell mass
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0000087
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: fresh specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000971
characteristics.characteristic: specimen with known storage state
characteristics.text: 129S4 Pdgfra::GFP
characteristics.characteristic: strain
characteristics.text: E3.5_ICM_2
characteristics.characteristic: title