organism: Danio rerio
description: Protocols: Zebrafish PGCs were isolated via FACS after embryo dissociation. The embryos were grown until the desired stage at 28.5C and collected in synchronous batches. Cell dissociation occurred via pipetting thoroughly up and down the pooled embryos until the solution appeared homogeneous. Yolk excess was removed by two rounds of centrifugation at 300 x g (5 minutes). Cells were passed through a 50 μm mesh and loaded into a FACS Aria II machine for cell sorting. Morpholino antisense oligos were used in order to inhibit Tdrd7 translation during zebrafish development. Stock morpholinos were diluted in phenol red and about 0.3 pM were injected into the yolk of a fertilised zebrafish embryo. As experimental control, a group of embryos were injected with morpholinos having 5 mismatches for the target Tdrd7 transcript. A rescue experiment was performed with Tdrd7 MO resistant RNA from tdrd7 transcript. The RNA was in-vitro transcribed and co-injected in once-cell embryo to rescue MO-mediated Tdrd7 KD. Cells were lysed in presence of RNAse inhibitor (0.2 U/μl) and cDNA generated from the cell lysate. cDNA was generated with the Takara SMART-Seq® v4 Ultra® Low Input RNA Kit, as from manufacturer's instruction. In brief, reverse transcription was carried out in presence of the SMART-Seq CDS Primer II A and SMARTScribeTM Reverse Transcriptase (Takara Bio Europe, 634889, France). Library amplification occurred according to protocol recommendations and the amplified cDNA purified via the Agencourt AMPure XP beads kit (Beckman Coulter, A63880, USA).
attributes.tag: ENA first public
attributes.value: 2020-12-24
attributes.tag: ENA last update
attributes.value: 2020-12-02
attributes.tag: External Id
attributes.value: SAMEA7678628
attributes.tag: INSDC center alias
attributes.value: MRC London Institute of Medical Sciences and Faculty of Medicine, Imperial College, London, UK
attributes.tag: INSDC center name
attributes.value: MRC London Institute of Medical Sciences and Faculty of Medicine, Imperial College, London, UK
attributes.tag: INSDC first public
attributes.value: 2020-12-24T04:05:28Z
attributes.tag: INSDC last update
attributes.value: 2020-12-02T17:13:26Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9857:PGC 5mm rep2
attributes.tag: age
attributes.value: 24
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: primordial germ cell
attributes.tag: common name
attributes.value: zebrafish
attributes.tag: developmental stage
attributes.value: pharyngula prim-5
attributes.tag: genotype
attributes.value: tg(buc:gfp)
attributes.tag: sample name
attributes.value: E-MTAB-9857:PGC 5mm rep2
attributes.tag: strain
attributes.value: AB