title: MAH183A609_S78
organism: Monodelphis domestica
description: Protocols: Oocytes and pre-implantation opossum embryos were harvested from time mated natural ovulation females. Oocytes were recovered from oviducts of females that were mated to sterile males. Embryos, E1.5 (36h.p.c), E2.5 (60h.p.c), E3.5 (84h.p.c), E4.5 (108h.p.c), E5.5 (132h.p.c), E6.5 (156h.p.c) and E7.5 (180h.p.c) pre-implantation opossum embryos were recovered from uterus into PBS-A. Harvested embryos were kept on ice in a petri dish with PBS-A while being processed. Individual embryos (ranging from E1.5 - E7.5) were transferred to new glass bottom 35mm petri dish with fresh PBS-A. Individual embryos were transferred to glass (1.5 glass) bottom 35mm petri dish with 14mm bottom well. Under the dissection microscope, the shell coat around individual embryos were removed using needles (30G) attached to an one ml syringe. Subsequently the mucin coat and zona pellucida were removed by incubation of the embryos in a pre-warmed pronase solution (0.5% Protease in PBS- A, Sigma) on 37 deg. C hot plate for 4 to 8 minutes, depending on its thickness of the mucin coat and on the stage of the embryos. Single cells, from E1.5 to E4.5 embryos, were picked manually with and rinsed in another glass bottom small petri dish with PBS-A. Rinsed cell was picked up with 1ul of PBSA and transferred to nonstick RNase and DNase free 0.5ul Eppendorf under the microscope. Eppendorf with sample was kept on dry ice and latter samples were stored in -80 deg. C. E5.5 to E7.5 embryos were first incubated with 0.5% trypLE (Gibco) in PBS for around 3 to 6 min at 37°C on hot plate, prior to gentle dissociation into single cells by agitation. Single cells were picked up manually and transferred to a fresh glass bottom petri dish with PBS-A. Samples were again stored in -80 deg. C for cDNA preparation. see 'nucleic acid library construction protocol Amplified double-stranded cDNA from single cells was prepared using SMART-Seq v4 Ultra Low Input RNA Kit and followed manufacturer's instructions (Cat no - 634891, Clontech Laboratories). Single cells cDNA was subjected to 17 to 18 cycles of PCR amplification. E2.5, E3.5 and E4.5 whole embryos cDNA underwent 14 to 15 cycles amplification; E5.5 and E6.5 embryos, 10 - 11 cycles; E7.5, 7 - 8 cycles. cDNA was analysed in the Bioanalyzer using Agilent's High sensitivity DNA Kit. Amplified double-stranded cDNA samples that had passed quality control checks were subsequently used to make libraries. A minimum of 1 ng of cDNA was used for the preparation of libraries using the Ovation Ultralow Library system V2 1-96 (Cat. no. - 0347) following the manufacturer's guidelines for library preparation.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-05-20T04:05:54Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-01-21T22:48:26Z
attributes.tag: External Id
attributes.value: SAMEA5231997
attributes.tag: INSDC center name
attributes.value: 1 - Duke-NUS Medical School 2 - Francis Crick Institute, UK
attributes.tag: INSDC first public
attributes.value: 2020-05-20T04:05:54Z
attributes.tag: INSDC last update
attributes.value: 2019-01-21T22:48:26Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7515:MAH183A609_S78
attributes.tag: age
attributes.value: 5.5
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: gray short-tailed opossum
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: individual
attributes.value: E38
attributes.tag: inferred cell type
attributes.value: pre / early-lineage
attributes.tag: sample name
attributes.value: E-MTAB-7515:MAH183A609_S78
attributes.tag: scientific_name
attributes.value: Monodelphis domestica
attributes.tag: sex
attributes.value: female