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Status |
Public on Oct 30, 2024 |
Title |
C. elegans, E. ulmoides extract, biol rep 5 |
Sample type |
SRA |
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Source name |
whole animal
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Organism |
Caenorhabditis elegans |
Characteristics |
time: Day 12 tissue: whole animal strain: N2 genotype: WT treatment: 30 ug/mL E.ulmoides extract and DMSO(0.05%)
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Treatment protocol |
The dried TCM plants (20 g each) Cuscuta chinensis and Eucommia ulmoides were extracted with a mixture of t-butyl methyl ether, methanol (50:50, volume 75 mL) as well as with 100% methanol (volume 75 mL). The extracts were dissolved in Dimethyl sulfoxide (DMSO) at a stock concentration of 60 mg/mL and then added to the NGM agar plates as well as the feeding bacterial strains at a final concentration of 30 μg/mL; DMSO (0.05%) was used in the control feeding bacteria and NGM agar. Synchronized, untreated L4 larvae were transferred to the NGM agar plates that had been previously prepared with 2 mg/mL carbenicillin. Finally, 100 µM 5-fluorodeoxyuridine (FUdR) was added. The nematodes were incubated at 22 °C until the 12th day of adulthood.
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Growth protocol |
The wild-type N2 Bristol C. elegans strain was maintained and cultured on NGM agar plates seeded with the E. coli strain OP50 as a food source. Synchronized worms were regularly generated by treating young adults with a 3% sodium hypochlorite solution until eggs were isolated. The obtained eggs were incubated with overnight shaking in M9 buffer, and the hatched L1 larvae were transferred to new NGM plates the following day. Two days later, the nematodes reached the fourth larval stage (L4) and were transferred to the respective treatment plates.
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Extracted molecule |
total RNA |
Extraction protocol |
On the 12th day of adulthood, the worms were collected and transferred to lysis tubes (around 3000-4000 worms per tube) with steel beads and stored at -80 °C. The RNA extraction proceeded according to the kit's manufacturer (innuSPEED Tissue RNA Kit by Analytik Jena Germany). The Turbo DNA-free kit from Thermo Fischer Scientific (Germany) was used at the end to degrade any possible DNA within the samples. The RNA content of each sample was measured with a Nanodrop and the condition of the RNA was checked with gel electrophoresis. The preparation of RNA library and transcriptome sequencing was conducted by Novogene Co., LTD (Beijing, China). Briefly, a total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform, and paired-end reads were generated.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
Eu11
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Data processing |
Raw reads filtering: Raw reads are filtered to remove reads with adapter contamination or reads with low quality. Only clean reads were used in the downstream analyses. The filtering process is as follows: (1) Discard reads with adaptor contamination. (2) Discard reads when uncertain nucleotides constitute more than 10% of either read (N > 10%). (3) Discard reads when low quality nucleotides (base quality less than 20) constitute more than 50% of the read. Mapping To Reference Genome Alignments were performed with HISAT2 to the reference. The analysis of the read counts data was conducted with the R-based tool DEBrowser, using the Bioconductor package eEdgeR for the differential expression analysis with a trimmed mean of M-values (TMM) normalization and filtering out genes, whose counts per million (CPM) was lower than one. The genes/transcripts with a p-value (adjusted by DEBrowser with the Benjamini Hochberg procedure) below 0.05 and fold change higher than 1.5 or lower than 0.667 were considered differentially expressed genes (DEGs). The DEG data were further analyzed using the online tool DAVID (Database for Annotation, Visualization and Integrated Discovery), to create a list of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways Assembly: Genome assembly WBcel235 Supplementary files format and content: The excel file Matrix_FPKM.xlsx contains all FPKM values. The gene identifiers are listed one per row according to the genome assembly WBcel235 and were annotaded according to Wormbase. The samples are displayed in columns.
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Submission date |
Aug 09, 2024 |
Last update date |
Oct 30, 2024 |
Contact name |
Nadine Saul |
Organization name |
Humboldt-Universitaet zu Berlin
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Department |
Department of Biology
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Street address |
Philippstr. 13, House 22
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City |
Berlin |
ZIP/Postal code |
10115 |
Country |
Germany |
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Platform ID |
GPL26672 |
Series (1) |
GSE274478 |
Transcriptomics of C. chinensis and E. ulmoides treated aged C. elegans |
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Relations |
BioSample |
SAMN43102837 |
SRA |
SRX25655447 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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