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ERX4516253: NextSeq 500 sequencing; RNA-seq of NvNcol3::mOrange2 positive and NvNcol3::mOrange2 negative cells from Nematostella vectensis
1 ILLUMINA (NextSeq 500) run: 60.2M spots, 4.5G bases, 1.7Gb downloads

Design: RNA-seq of NvNcol3::mOrange2 positive and NvNcol3::mOrange2 negative cells from Nematostella vectensis
Submitted by: Sars Centre for Marine Molecular Biology, University of Bergen (Sars Centre for Marine Molecular Biology, Universi)
Study: RNA-seq of NvNcol3::mOrange2 positive and NvNcol3::mOrange2 negative cells from Nematostella vectensis
show Abstracthide Abstract
NvNcol3::mOrange2 is a stable transgenic line that labels cnidocytes(stinging cells) of the sea anemone Nematostella vectensis (Nakanishi et al., Development 2012). Two week old primary polyps were dissociated and the NvNcol3::mOrange2 positive and negative cells were enriched by FACS.
Sample: Ncol3mOrange2 Pos R2
SAMEA7301426 • ERS5059985 • All experiments • All runs
Library:
Name: Ncol3mOrange2 Pos R2_s
Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: Oligo-dT
Layout: SINGLE
Construction protocol: FACS sorting were performed as previously described (Torres-Mendez et al. 2019). Briefly, animals were dissociated in 0.25% Trypsin (Gibco, 27250018) in Ca- Mg- free Nematostella media (CMFNM) (154 mM NaCl, 3.6 mM KCl, 2.4 mM Na2SO4, 0.7 mM NaHCO3) supplemented with 6.6 mM EDTA , pH 7.6-7.8. Cells were centrifuged at 800 g for 10 minutes, resuspended in ice cold 0.5% BSA in CMFNM (pH 7.6-7.8), filtered through a 40 μM filter and stained with Hoechst 33342 (Thermo Fisher Scientific, 62249) at a 60 μg/ml at RT for 30 minutes. Samples were then diluted 1:1 with ice cold 0.5% BSA/CMFNM and stained with 60 μl/ml 7-AAD (BD, 559925) for > 20 minutes on ice. Sorting was carried out on a BD FACSAria II with a 100 μm nozzle. RNA extraction was performed as previously published (Torres-Mendez et al. 2019). Cells were sorted directly into 0.5% BSA/ CMFNM at 4°C and centrifuged at 800 g for 10 minutes at 4°C. Most of the liquid was removed and 3 volumes of TRIzol LS reagent (Invitrogen, 10296028) was added. Samples were vortexed extensively and incubated at RT for 5 minutes before being flash frozen and stored at -80°C. The samples were processed using Direct-zol RNA MicroPrep columns (Zymo Research, R2060) including on column DNase digestion. cDNA was prepared from 400 pg of total RNA using the Smart-Seq 2 method with 16 pre-amplification PCR cycles (Picelli et al. 2014). NGS libraries were prepared using the home-made tagmentation-based method (Hennig et al. 2018). Briefly, 125 ng of cDNA was tagmented using home-made Tn5 loaded with annealed linker oligonucleotides for 3 minutes at 55°C. Reactions were inactivated by adding 1.25 ml of 0.2% SDS and incubation for 5 minutes at RT. Indexing and amplification was done using the KAPA HiFi HotStart PCR kit (Sigma-Aldrich, KK1508) with Index oligonucleotides (sequences were adapted from Illumina).
Experiment attributes:
Experimental Factor: cell type: NvNcol3::mOrange2 Positve cells
Runs: 1 run, 60.2M spots, 4.5G bases, 1.7Gb
Run# of Spots# of BasesSizePublished
ERR458062360,187,1634.5G1.7Gb2021-07-02

ID:
15120860

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