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Status |
Public on Dec 31, 2022 |
Title |
105-G-Tb-3 |
Sample type |
SRA |
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Source name |
tentacles
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Organism |
Hydra vulgaris |
Characteristics |
strain: Hm_105 tissue: original tentacle proximal halves genotype: wild type treatment: mock RNAi
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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.
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Growth protocol |
standard Hydra culture conditions
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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).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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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
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Submission date |
Dec 17, 2021 |
Last update date |
Dec 31, 2022 |
Contact name |
Panagiotis Papasaikas |
E-mail(s) |
panp80@gmail.com
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Phone |
+41791074582
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Organization name |
Friedrich Miescher Institute
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Street address |
66 Maulbeerstrasse
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City |
Basel |
ZIP/Postal code |
CH-4058 |
Country |
Switzerland |
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Platform ID |
GPL19551 |
Series (1) |
GSE191177 |
The transcription factor Zic4 promotes tentacle formation and prevents epithelial transdifferentiation in Hydra |
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Relations |
BioSample |
SAMN24174089 |
SRA |
SRX13443446 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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