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Sample GSM1961244

Query DataSets for GSM1961244

Status

Public on Dec 04, 2015

Title

Other Cells 1

Sample type

SRA

Source name

FACS sorted mix of differentiated cells

Organism

Ephydatia fluviatilis

Characteristics

genotype/variation: wild type
tissue: whole juvenile sponges
cell type: Other Cells

Treatment protocol

for de novo transcriptome sequencing, 450 μg of total RNA was extracted from sponges at different stages using an IsogenLS kit (Nippon Gene Co., Ltd). For RNA-seq on cell fractions, Choanocytes were labelled following a protocol slightly modified from (20): stage 5 sponges (7 days old) were incubated in 2 μg.ml-1 Hoechst 33342 (in M-medium) for 30 minutes. During the last 8 minutes of this incubation, sponges were fed FluoSpheres (carboxylate-modified microspheres, 0.2 μm in size, yellow-green fluorescent (505/515 nm) (Molecular Probes, Eugene, OR, USA)), at a concentration of 1μl ml-1 in M-medium. Then the sponges were washed 3 times for 5 minutes each with M-medium. Archeocytes were labelled as follows: sponges were cultivated in M-medium with 2 μg μl-1 Rhodamine 123 in the dark until they reached stage 1-2. Rho 123 is a fluorescent dye that accumulates in the mitochondria due to their transmembrane potential (53), and we found that it was also incorporated into the abundant vitelline platelets present in the archeocyte cytoplasm (probably because the vitelline platelets contain mitochondria (54)). Then sponges were washed 3 times for 1 hour each in normal M-medium and incubated in 2 μg ml-1 Hoechst 33342 (in M-medium) for 30 minutes and washed 3 times for 5 minutes each in M-medium. Cells belonging to the “other cells” fraction were collected from stage 1-2 sponges labelled with Rho123 and Hoechst 33342, and fed FluoSpheres as described above. Gemmules used in these experiments were isolated from the same mother sponge. Before FACS sorting, sponges were rinsed with Ca2+, Mg2+-free medium (CMFM: Na2SiO3, 22.48 g l-1; NaHCO3, 13.34 g l-1; KCl, 0.37 g l-1) twice and scraped and pipetted to dissociate cells in 2 ml of CMFM. Dissociated cells were filtered using a 35-μm nylon mesh (cell-strainer cap tube, Beckton-Dickinson Falcon, Franklin Lakes, NJ, USA) to remove spicules, and then maintained on ice. Dissociated cells were stained with 2 μg ml-1 propidium iodide (PI, Dojindo, Kumamoto, Japan) on ice for 5 min and immediately subjected to cell sorting using a BD FACS Aria 2 (BD Bioscience, San-Jose CA, USA). We empirically determined thresholds to collect Hoechst-positive, PI-negative viable cells. Archeocytes were isolated as large cells displaying Rho123 fluorescence (40,000 cells collected per biological replicate), choanocytes as small cells displaying FluoSphere fluorescence (100,000 cells collected per biological replicate), and “other cells” as cells having no fluorescence and SSC/FSC values not overlapping with those of choanocytes or archeocytes (100,000 cells collected per biological replicate). The cell collection device was maintained at 4°C throughout the procedure. The collected samples were centrifuged at 13,000 rpm, 4°C, 10 minutes, and the supernatant was removed. Samples were frozen at -80°C.

Growth protocol

Gemmules were isolated and juvenile sponges were cultivated from them in M-medium

Extracted molecule

total RNA

Extraction protocol

RNeasy Micro kit (Qiagen, Hilden, Germany), following the instructions of the manufacturer. DNase digestion was performed. For each replicate, 10 ng of total RNA were used as the starting material
SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech Takara Bio Inc., Mountain View, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

For de novo transcriptome assembly : Oases software with default parameters produced 133,045 transcripts grouped into 39,484 loci with an average length of 1,216 nucleotides (not shown). Visual inspection of this assembly revealed that many predicted isoforms were in fact transcripts differing by only a few substitutions or gaps. Therefore, we decided to select a single transcript for each locus based on criteria of size and fold-coverage, using the publicly available Oases-to-csv python script (https://code.google.com/p/oases-to-csv/downloads/list). Then we kept only the orfs over 270 nucleotides long using Transdecoder (http://transdecoder.sourceforge.net/), and ended up with 17,419 transcripts with an average length of 1,152 nt (provided in File S1). This is of the same order of magnitude as the transcriptome of the close species Ephydatia muelleri, recently released on the Compagen platform (http://www.compagen.org/), containing 21,804 transcripts over 270 nt long with an average length of 1,242 nt.
Gene expression levels were calculated using RSEM-1.2.4 (55): reads from each duplicate were mapped on the 17,419 reference transcripts using default Bowtie parameters (parameter bowtie-m 200, seed length of 25 nt). Resulting expected counts were analysed using EdgeR 3.0.0 (56) (which assumes that read counts have a negative binomial distribution): duplicate reproducibility was first confirmed by principal component analysis (Fig. S1); reads counts were TMM normalised; the common dispersion was calculated for each condition
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

Submission date

Dec 03, 2015

Last update date

May 15, 2019

Contact name

Noriko Funayama

Organization name

Kyoto University

Department

Biophysics

Lab

Molecular Developmental Biology