Platynereis dumerilii
Protocols: Wild-type P. dumerilii adults were spawned and larvae were incubated in filtered natural seawater, at 18°C, for 47 hours, to reach the desired stage. For cell dissociation, ca 100 47hpf P.dumerilii larvae were picked and incubated for 5 minutes in 0.5% Pronase (Roche cat # 10165921001), 1% sodium thioglycolate (Sigma T0632) diluted in calcium- and magnesium-free artificial sea water. After this treatment, the separated brains (epispheres) were picked, rinsed in 1xPBS, and incubated for 1 minute in 150 ug/ml Liberase (Roche, cat # 05401119001) in 1xPBS. Epispheres were rinsed twice in 1xPBS, transferred to a non-stick eppendorf tube and triturated by pipetting. Dissociated cells were washed twice in 200 ul of 1xPBS and concentrated by centrifuging (1 min, 200xg). Cells were resuspended in 20 ul of 1xPBS, of which 5 ul was loaded on the capture chip (C1 IFC for mRNA-seq for 10-17 um cells, Fluidigm cat # 100-5760). Cell capture, lysis, reverse transcription and cDNA amplification was performed on the C1 IFC for mRNA-seq (10-17 μm, Fluidigm cat # 100-5760) on Fluidigm C1 Single-Cell Auto Prep System. We used SMARTer Ultra Low Input RNA Kit for reverse transcription and the ADVANTAGE-2 PCR kit (Clontech cat # 634832) for PCR. We added 1 μl of 1:4000 dilution of ERCC spike-in RNA (Ambion, cat # 4456740) to the cell lysis mix. Sequencing libraries were prepared using the Nextera XT DNA Sample Preparation kit (Illumina cat # FC-131-1096) ). The samples in this study were derived from altogether 3 IFC-s (termed C3, C4, C5 in the batch information). The samples from IFCs C3 and C4 were pooled into one sequencing library. The samples from IFC C5 were pooled into a separate sequencing library.
ENA-FIRST-PUBLIC
2014-09-29T17:05:35Z
ENA-LAST-UPDATE
2018-03-08T20:19:51Z
External Id
SAMEA2757589
INSDC center name
European Bioinformatics Institute, European Molecular Biology Laboratory (EMBL-EBI)
INSDC first public
2014-09-29T17:05:35Z
INSDC last update
2018-03-08T20:19:51Z
INSDC status
public
Submitter Id
E-MTAB-2865:C5x04-nc
broker name
ArrayExpress
common name
Dumeril's clam worm
developmental stage
48 hpf
organism part
episphere
sample name
E-MTAB-2865:C5x04-nc
scientific_name
Platynereis dumerilii
single cell well quality
no cell