title: NextSeq 550 paired end sequencing; RNA-seq of zebrafish primordial germ cells and somatic cells during early embryogenesis
library_construction_protocol: 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. 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).