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Sample GSM5740459 Query DataSets for GSM5740459
Status Public on Dec 31, 2022
Title 105-G-Tb-3
Sample type SRA
 
Source name tentacles
Organism Hydra vulgaris
Characteristics strain: Hm_105
tissue: original tentacle proximal halves
genotype: wild type
treatment: mock RNAi
Treatment protocol 20-30 animals per RNAi treatment were incubated at 4 °C for 1 hour and then transferred into an electroporation cuvette (Gene Pulser / Micro Pulser Electroporation Cuvette 0.4 cm gap; Bio-Rad or electroporation cuvettes 4 mm gap; VWR) and washed twice with chilled MiliQ water. Residual water was removed and 200 μL of the siRNA (siRNAs were ordered as 20 bp RNA duplexes from Integrated DNA Technologies (IDT)) solution was added to each cuvette. Each siRNA was diluted in ddH2O to a final concentration of 1.35 μM per treatment. Animals were incubated with the siRNA solution for 5 minutes. After relaxation of the animals, two pulses (150 V range) were applied for 50 ms (Gene Pulser II with RF module; Bio-Rad). After electroporation, 500 μL of ice-cold recovery medium (80 % HM, 20 % v/v dissociation medium) was added to the animals. Animals were carefully transferred into a new Petri dish filled with pre-chilled recovery medium. The next day, the animals were transferred into fresh HM. Three electroporations were performed in this manner with 1 day for recovery in between successive rounds.
Growth protocol standard Hydra culture conditions
Extracted molecule total RNA
Extraction protocol Original and ectopic tentacles were collected 7 days after the third electroporation from mock, Zic4, Sp5, and Zic4/Sp5 RNAi animals. When possible, the proximal and distal tentacle halves were separated (original tentacles of Zic4 RNAi and Sp5 RNAi). Corresponding samples were pooled in the lysis buffer, frozen on dry ice and stored at -80 °C for RNA extraction. The RNA was extracted with the Single Cell RNA Purification Kit (Norgen), according to the manufacturer’s instructions.
cDNA amplification was performed using the SmartSeq2 approach as per the original protocol (31). Full length cDNA was processed for Illumina sequencing using Tagmentation with an in-house purified Tn5 transposase (32): 1ng of amplified cDNA was tagmented in TAPS-DMF buffer (10mM TAPS pH8.5, 5mM MgCl2, 10% DMF), at 55C for 7min. Tn5 was then stripped using SDS (0.04% final) and tagmented DNA was amplified using Phusion High-Fidelity DNA Polymerase (ThermoFisher). PCR was performed in the Phusion HF buffer, with a first extension at 72 °C for 3min, followed by 10 cycles of amplification (95 °C – 30 s, 55 °C –30 s, 72 °C – 30s). Commercial Nextera XT indexes were used for the PCR amplification (1/5 dilution).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Data processing Final libraries were sequenced on a HiSeq2500 (50cycles single-end) and demultiplexing performed using a standard Illumina bcl2fastq2 pipeline.
Reads were aligned against the hydra vulgaris NCBI genome guided by transcriptome annotation (NCBI Hydra vulgaris assembly Hydra_RP_1.0, NCBI Hydra vulgaris annotation release 102).
Alignments were performed using STAR version 2.5.0 with the following command line parameters: --outSJfilterReads Unique --outFilterType BySJout --outFilterMultimapNmax 5 --alignSJoverhangMin 8 --alignSJDBoverhangMin 4 --outFilterMismatchNoverLmax 0.1 --alignIntronMin 20 --alignIntronMax 1000000 --outFilterIntronMotifs RemoveNoncanonicalUnannotated --seedSearchStartLmax 50 --twopassMode Basic --genomeChrBinNbits 12 --genomeSAsparseD 2 --quantMode GeneCounts.
Raw (exonic) gene counts as determined by the STAR GeneCounts quantification mode
Genome_build: NCBI Hydra_RP_1.0
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
 
Submission date Dec 17, 2021
Last update date Dec 31, 2022
Contact name Panagiotis Papasaikas
E-mail(s) panp80@gmail.com
Phone +41791074582
Organization name Friedrich Miescher Institute
Street address 66 Maulbeerstrasse
City Basel
ZIP/Postal code CH-4058
Country Switzerland
 
Platform ID GPL19551
Series (1)
GSE191177 The transcription factor Zic4 promotes tentacle formation and prevents epithelial transdifferentiation in Hydra
Relations
BioSample SAMN24174089
SRA SRX13443446

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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