GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM847463

Query DataSets for GSM847463

Status

Public on Apr 25, 2012

Title

Asexual Non-irradiated [paired end]

Sample type

SRA

Source name

Asexual strain of S. mediterranea, Non-irradiated

Organism

Schmidtea mediterranea

Characteristics

strain: Asexual
treatment: Non-irradiated
cell type: N/A

Treatment protocol

Sexual and asexual animals were gamma-irradiated with a cesium source with 90 Gy treatments. D. tigrina animals were ordered from Carolina Biological and RNA was isolated immediately upon arrival.

Growth protocol

Sexual and asexual S. mediterranea animals were maintained at 22°C in ddH2O supplemented with 1.6 mM NaCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 0.1 mM MgCl2, 0.1 mM KCl, 1.2 mM NaHCO3 and fed homogenized organic beef liver. FACS purification of X1, X2 and Xins cells was carried out as follows. Briefly, planarians were cut into small pieces with a sterile scalpel on ice in cold calcium, magnesium-free medium (CMF) (15 mM HEPES, 400 mg/L NaH2PO4, 800 mg/L NaCl, 1200 mg/L KCl, 800 mg/L NaHCO3, 240 mg/L glucose at pH 7.3) and then washed twice with CMF. The fragments were then soaked in CMF containing 0.25% (w/v) Trypsin (GIBCO) and 1% BSA (Fisher Scientific) at 4°C for 18 hours to maximize penetration of the enzyme with little trypsin activity. The fragments were then rocked for 20 minutes at 20°C and completely dissociated into single cells by gentle pipetting. The cell mixture was then sequentially filtered through 40-µm and 20-µm nylon filters (Millipore). Cells were collected by centrifugation, resuspended in fresh CMF supplemented with 1% BSA and 0.5 mg/mL Calcein AM (Sigma), and incubated at room temperature for 20 minutes. Cells were collected and resuspended in CMF supplemented with 1% BSA, 18 mg/mL Hoechst 3342 (Sigma), and 2 mg/mL propidium iodide (Sigma). The samples were then analyzed with a LSR II flow cytometer (Becton-Dickinson).

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from sexual and asexual strains of S. mediterranea before and after irradiation using Trizol. Total RNA (5ug) was used to prepare the transcriptome library using the mRNA-seq sample library preparation kit (Illumina) following the manufacturers instructions. Briefly, mRNA was purified using magnetic oligo-dT beads and fragmented. First strand synthesis of cDNA was done using SuperscriptII reverse transcriptase followed by RNase H treatment and second strand cDNA synthesis was performed using DNAPol1. Paired end adaptors (Illumina) were ligated to 5’ and 3’ ends of DNA fragments followed by PCR amplification using adaptor specific primers. PCR amplified products of 300 bp were gel purified, quantitated by Nanodrop and Bioanalyzer and sequenced on an Illumina GAIIx.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Description

Asexual strain of S. mediterranea, Non-irradiated

Data processing

The S. mediterranea draft genome assembly (version 3.1) consists of ~900 Mb distributed over 43,294 supercontigs (http://genome.wustl.edu/genomes/view/schmidtea_mediterranea/). To facilitate the analysis the supercontigs were sorted by decreasing size and concatenated into nine pseudochromosomes of ~100 Mbp each with 100 'N' residues separating each contig. These pseudochromosomes are contained in the Smed_pseudochromosomes.fasta file. Reads were aligned to the pseudochromosomes using bowtie-0.12.1 and tophat-1.1.1 using default parameters. To assemble the D. tigrina transcript contigs, a total of 23,532,916 D. tigrina reads of 40 bp were used with Velvet 0.3 using k=15. This resulted in 14,514 contigs with an average length of 140 bp and an average coverage of 8.7X.

Submission date

Dec 09, 2011

Last update date

May 15, 2019

Contact name

Brenton R Graveley

E-mail(s)

graveley@uchc.edu

Phone

860-679-2090

Organization name

University of Connecticut Health Center