description: Protocols: Embryos were collected by non-surgical uterine flush between E5.0 and E7.0. Marmoset blastocysts were staged based on embryonic day, diameter and blastocoel formation. Where appropriate, zona pellucidae were removed using acid Tyrode's solution (Sigma) and embryos were subjected to immunosurgery as previously described (Solter and Knowles, Proc Natl Acad Sci USA 72:5099-102; Nichols et al. Cell 95:379-91). Briefly, blastocysts were incubated for 15min with a custom rabbit polyclonal anti-marmoset antibody 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 dissociated from residual trophectoderm with a narrowly fitting glass pipette. For postimplantation embryos the epiblast was isolated by manual dissection. Extraembryonic ectoderm was first removed with a wide mouth-controlled pipette, and the visceral endoderm was then mechanically separated by drawing the embryonic portion through a narrow glass aperture. Dissociation of morulae, ICMs and postimplantation epiblast was performed in a 1:1 mixture of 0.025% trypsin plus EDTA (Invitrogen) and 0.025% trypsin (Invitrogen) plus 1% chick serum (Sigma). Morulae and early ICMs were incubated for 5min at 37 C, and E4.5 ICMs and postimplantation epiblasts were incubated for 10min. The explants were washed in N2B27 supplemented with 10µM HEPES and 1.5mg/ml BSA (Gibco) and dissociated in a small drop of medium using blunted microcapillaries with an inner diameter large enough to accommodate approximately 2-3 cells. Cells were transferred immediately into 4.5ul of single-cell lysis buffer and snap frozen on dry ice. cDNA synthesis was performed using whole transcriptome preamplification (Tang et al. Nat Methods 6:377-382, Tang et al. Nat Protoc. 5:516-535). cDNA was preamplified 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 20 C 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 optimized 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2015-11-19T17:11:57Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T20:17:07Z
attributes.tag: External Id
attributes.value: SAMEA3292277
attributes.tag: INSDC center name
attributes.value: European Molecular Biology Laboratory
attributes.tag: INSDC first public
attributes.value: 2015-11-19T17:11:57Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T20:17:07Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2959:CM_Mid_ICM_R2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: white-tufted-ear marmoset
attributes.tag: developmental stage
attributes.value: mid-stage blastocyst
attributes.tag: organism part
attributes.value: embryo
attributes.tag: sample name
attributes.value: E-MTAB-2959:CM_Mid_ICM_R2
attributes.tag: scientific_name
attributes.value: Callithrix jacchus