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Sample GSM929778

Query DataSets for GSM929778

Status

Public on Jun 01, 2012

Title

Stem Cells

Sample type

SRA

Source name

FACS purified X1s

Organism

Schmidtea mediterranea

Characteristics

tissue: Stem cells, neoblasts, X1s

Treatment protocol

To FACS purify stem cells, animals were dounced in eppendorf tubes in cold Calcium, Magnesium-Free (CMF) media, and passed through a 20um filter. Cells were incubated with 25ug/ml Hoechst 33342 for 30min, pelleted at 400g, resuspended in CMF, and sent through a BD FACSaria. Cells were sorted by side scatter, forward scatter, then Hoechst red vs. Hoechst Blue. High DNA-containing cells that were absent in the irradiated controls, were collected as the stem cell fraction. The side population of low-DNA cells, which are stem cell progeny, were simultaneously collected in a separate tube. Whole, 7-day lethally irradiated planarians were used as stem cell lineage negative, differentiated tissue samples. Approximately 1.3 million cells, collected over 7 separate sorts, were pooled for each sample, and RNA extracted.

Growth protocol

The clonal strain of Schmidtea mediterranea, CIW4, was grown in a milli-Q-based salt solution on beef liver. Organisms were starved for 1 week, then processed for FACS purification.

Extracted molecule

total RNA

Extraction protocol

All 3 tissue samples were RNA extracted in Trizol reagent, and RNA quality was analyzed on a bioanalyzer. RNA samples were library prepped using Illumina's Truseq Library preps, which included polyA selection.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Data processing

Base calls were made using the Illumina pipeline v. 1.6 for Illumina GAIIx and v. OLB 1.9 for HiSeq2000
All RNAseq was pooled and a de novo transcriptome was made using the program Trinity with default settings.
BPKG_transcriptome was deposited into NCBI TSA database as reference number: SUB086338; BioProject ID: PRJNA163163
Genome_build: BPKG_transcriptome.nt
Supplementary_files_format_and_content: RPKM quantification for all 3 cellular compartments used in this study, as well as top-hit BLAST annotation to mouse homologs.

Submission date

May 10, 2012

Last update date

May 15, 2019

Contact name

Bret J Pearson

E-mail(s)

pearsobr@ohsu.edu

Organization name

Oregon Health and Science University

Department

Pediatrics