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Sample GSM2187964

Query DataSets for GSM2187964

Status

Public on Dec 01, 2016

Title

Smed_Sexual_Virgin_4

Sample type

SRA

Source name

virgin, sexuallly mature adult

Organism

Schmidtea mediterranea

Characteristics

biotype: sexual
day: not applicable
replicate: 4

Treatment protocol

None

Growth protocol

Animals were maintained in 1x Montjuic water at 20˚C in the dark and fed homogenized beef liver once or twice per week. Cultures subjected to intensive feeding regimens were supplemented with 100 µg/mL gentamicin sulfate Gemini Bioproducts, #400-100P). The asexual clonal strain CIW-4 (Newmark and Sanchez Alvarado 2002) was propagated via successive rounds of amputation and regeneration. Sexually reproducing S. mediterranea stocks were descendants of animals collected in Corsica by Dr. Maria Pala in 1999. Egg capsules were collected daily from outbred cohorts of sexually mature adults cultured at low density (6-8 animals per 400 mL culture), and were stored in dated petri dishes at 20˚C in constant darkness until use. The collection date was considered to be 1 day post-egg capsule deposition. To maintain optimal fertility levels, sexually mature animals used for egg capsule collections were replaced every 3-4 months with either juveniles (6-8 weeks post hatching) or adult regenerates (6-8 weeks post cut).

Extracted molecule

polyA RNA

Extraction protocol

Live embryos were dissected out of egg capsules in 1x Holfreter’s buffer (3.5 g/L NaCl; 0.2 g/L NaHCO3; 0.05 g/L KCl; 0.2 g/L MgSO4; 0.1 g/L CaCl2; 1.0 g/L dextrose, pH 7.0-7.5) for Stage (S) S2-S7 egg capsules, or 1x Montjuic water for S8 hatchlings. Yolk samples were obtained from 8 d egg capsules that contained neither spherical nor elongating embryos. Single embryos were transferred into microfuge tubes containing 200 µl TRIzol reagent (Thermo Fisher, item #15596-018), homogenized by pipetting, and stored at -80˚C. Single animal samples of intact C4 adults and virgin, sexually mature adults were homogenized in 1.0 mL TRIzol using an IKA Ultra Turrax T 25 Basic tissue disruptor prior to storage at -80˚C. Total RNA extraction was performed in 1.0 mL TRIzol per sample according to the manufacturer’s protocol, following the recommendations for working with small amounts of tissue.
PolyA-selected, single stranded RNA-Sequencing libraries were prepared for four biological replicates per stage using the Illumina TruSeq RNA Sample V2 kit (item # RS-122-2001 and RS-122-2002), starting with 500 ng total RNA per sample (C4, virgin sexual adult (SX), Y, S4, S5, S6, S7, S8), or 100 ng total RNA per sample (S2, S3).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Single intact virgin, sexually mature adult hermaphrodite

Data processing

Base calls were performed using CASAVA-1.8.2
Reads were aligned using bowtie with the following parameters: --best --strata -v 2 -m 5 against transcriptome smed_20140614 defined in GEO submission: GSE72389
Read counts to genes were tallied from the SAM files with a custom script. RPKM values were generated using the rpkm function from the edgeR library from Bioconductor in R.
Genome_build: Schmidtea_mediterranea_3.1
Supplementary_files_format_and_content: rpkm.txt contains tab-delimited RPKM vales for each sample.

Submission date

Jun 06, 2016

Last update date

May 15, 2019

Contact name

Chris W Seidel

E-mail(s)

seidel@phageT4.org

Phone

816 926 9054

Organization name

Stowers Institute