title: C2x58_1S
organism: Platynereis dumerilii
description: C2x58_1S Protocols: Wild-type P. dumerilii adults were spawned and larvae were incubated in filtered natural seawater, at 18oC, 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 um, 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 ul 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 5 IFC-s (termed C1, C2, C3, C4, C5 in the batch information). The samples from IFCs C1 and C2 were pooled into one sequencing library. This library was loaded on 2 lanes on Illumina HiSeq2000, obtaining 2 technical replicates per sample. 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2014-12-12T17:01:49Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T22:09:11Z
attributes.tag: External Id
attributes.value: SAMEA3175177
attributes.tag: INSDC center name
attributes.value: European Bioinformatics Institute, European Molecular Biology Laboratory (EMBL-EBI)
attributes.tag: INSDC first public
attributes.value: 2014-12-12T17:01:49Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T22:09:11Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2865_2:C2x58_1S
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: nervous system cell
attributes.tag: common name
attributes.value: Dumeril's clam worm
attributes.tag: developmental stage
attributes.value: 48 hpf
attributes.tag: individual
attributes.value: C2x58_1S
attributes.tag: organism part
attributes.value: episphere
attributes.tag: sample name
attributes.value: E-MTAB-2865_2:C2x58_1S
attributes.tag: scientific_name
attributes.value: Platynereis dumerilii