GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5590216

Query DataSets for GSM5590216

Status

Public on Sep 27, 2021

Title

DAPT6 6h rep6

Sample type

SRA

Source name

whole animals

Organism

Hydra vulgaris

Characteristics

treatment: DAPT
replicate: 6
tissue: basel
time point: 6h

Treatment protocol

Regularly fed animals were starved for 24 h and incubated in either 20 μM DAPT with 1% DMSO in Hydra medium or in only 1% DMSO in Hydra medium (control sample) for 48 h. DAPT and DMSO were renewed every 12 h. Animals were collected, and total RNA was isolated at three different time points: directly at the end of 48 h (0 h), 3 h after DAPT removal (3 h) and 6 h after DAPT removal (6 h). After 48 h incubation, DAPT was removed and replaced with 1% DMSO in Hydra medium for the samples 3 h and 6 h. About 25 animals were collected per sample

Growth protocol

Animals of the strain Hydra vulgaris (Basel) were grown in Hydra medium (0.1 mM KCl, 1 mM NaCl, 0.1 mM MgSO4, 1 mM Tris and 1

Extracted molecule

total RNA

Extraction protocol

RNA-seq libraries were prepared for six biological replicates for each experimental condition. cDNA libraries were synthesized from total RNA using the strand specific SENSE mRNA-Seq Library Prep Kit V2 for Illumina (Lexogen) and the Purification Module with Magnetic Beads (Lexogen). The samples were multiplexed and sequenced on three lanes on Illumina Hiseq2000 with a 100 bpsequencing strategy.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

The reads were demultiplexed. Illumina adapters and polyA sequences were trimmed and splice leader sequences (Stover and Steele, 2001) were removed from both forward and reverse reads. The tool ‘fastqfilter’ was used to ensure the paired nature of the filtered dataset, to filter out reads with a quality score lower than 20 and to exclude reads with a read length shorter than 30bp . Reads that contained ‘N’s were also removed from the dataset. Uploaded are the final reads.

Data processing

Illumina adapters and polyA sequences were trimmed and splice leader sequences (Stover and Steele, 2001) were removed from both forward and reverse reads. The tool ‘fastqfilter’ was used to ensure the paired nature of the filtered dataset, to filter out reads with a quality score lower than 20 and to exclude reads with a read length shorter than 30bp . Reads that contained ‘N’s were also removed from the dataset.
All forward and all reverse reads from all sequencing libraries were concatenated. The two resulting files were then used as input to the Trinity (version 2.8.4; Grabherr et al., 2011) de novo transcriptome assembler.
The assembly was run with the following parameters: -strand-specific library, in silico normalisation, -min_contig_length 300, -min_kmer_cov 1, no genome guided mode and no Jaccard Clip options.
The processed reads of the 36 RNA-seq libraries (timepoints 0, 3 and 6 h, DAPT and control samples, six replicates) were separately mapped to the de novo assembled transcriptome reference, within the Galaxy platform. The reads were mapped as strand-specific and with a maximum insert size of 800. RSEM (Li and Dewey, 2011) - with Bowtie2 (Langmead and Salzberg, 2012; Langmead et al., 2009) as alignment method- was used as the abundance estimation method.
The raw counts were used as input for differential expression (DE) analysis by DESeq2 (version 1.18.1).

Submission date

Sep 20, 2021

Last update date

Sep 27, 2021

Contact name

Jasmin Moneer

Organization name

LMU

Department

Biologie II

Lab

Boettger