Platynereis dumerilii
Protocols: Cell capture, lysis, reverse transcription and cDNA amplification was performed on the C1 IFC for mRNA-seq, 10-17 μm, Fluidigm cat # 100-5760 or C1 IFC for mRNA-seq for 5-10 um cells, Fluidigm cat # 100-5759) 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)). 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 500 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, animals were rinsed in 1xPBS, and incubated for 1 minute in 150 ug/ml Liberase (Roche, cat # 05401119001) in 1xPBS. After this, partially dissociated tissue was rinsed 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, or C1 IFC for mRNA-seq for 5-10 um cells, Fluidigm cat # 100-5759). The cDNA libraries were prepared using the Illumina Nextera XT DNA library preparation kit (Illumina # FC-131-1096) and dual barcoding with Nextera XT Index Kit (Illumina # FC-121-1012).
ENA first public
2017-09-17
ENA last update
2017-09-07
External Id
SAMEA104286427
INSDC center alias
Developmental Biology Unit, European Molecular Biology Laboratory (EMBL)
INSDC center name
Developmental Biology Unit, European Molecular Biology Laboratory (EMBL)
INSDC first public
2017-09-17T17:01:36Z
INSDC last update
2017-09-07T14:18:44Z
INSDC status
public
Submitter Id
E-MTAB-5953:C31x2401
age
48
broker name
ArrayExpress
common name
Dumeril's clam worm
developmental stage
larval stage
genotype
wild type genotype
inferred cell type
undifferentiated cell
organism part
whole organism
sample name
E-MTAB-5953:C31x2401
single cell well quality
single cell