|
| Status |
Public on Jul 05, 2021 |
| Title |
RAF_NM_4 [H01814] |
| Sample type |
SRA |
| |
|
| Source name |
midguts of 15 Drosophila melanogaster individuals
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Midguts
disease state: Cancer
diet: NM
feeding time: 13 days
|
| Treatment protocol |
We used a standard corn meal based medium for all stock flies and crossings. All assays were conducted on low-melt fly food containing 1.5% agar, 2% bacto yeast extract and 7% corn syrup (NM). The medium was boiled and supplemented with 0.1% propionic acid and 0.3% nipagin to prevent microbial growth. For dietary restriction (DR) the amount of bacto yeast was reduced to 0.1%. The recurrent diet consisted of an alternating regimen of four days DR followed by feeding on NM for three days.
|
| Growth protocol |
We used homozygous flies containing esg-Gal4, UAS-GFP, tubulin-Gal80ts on the 2nd chromosome, and UAS-Luciferase on the 3rd chromosome and crossed them to homozygous UAS-RAFgof flies to obtain the F1 generation with a temperature inducible tumor phenotype. As a control we crossed EGT;Luc2 to w1118 wildtype flies. All fly lines were raised on a cornmeal based standard medium at 20 °C. The F1 generation was shifted to 29 °C 5-7 days post eclosion to induce the expression of UAS-dependent genes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
DNA for 16S rRNA sequencing was extracted with the DNeasy Blood & Tissue Kit (Qiagen, #69504) following the manufacturer´s protocol for Pretreatment for Gram-Positive Bacteria, followed by the protocol for Purification of Total DNA from Animal Tissues. For RNA isolation 15 midguts per treatment were dissected in S2 medium, immediately transferred to a 2 ml screw cap tube containing 1 ml RNA Magic (Bio-Budget, #56-1000-100) and homogenized for 2 min at 3.25 m/sec using a bead mill homogenizer with 3 zirconium beads per tube. The homogenate was incubated for 5 min at RT and stored at -20 °C until further processing. Samples were thawed on ice for subsequent RNA isolation. 200 μl chloroform were added into each tube. Tubes were inverted (not vortexed) several times to provide homogenous mixing and incubated for 5 min at RT prior to 15 min centrifugation at 12.000 xg and 4 °C. The upper aqueous phase was transferred into a new tube (about 500 μl) and 500 μl 99% EtOH were added. The samples were centrifuged for 45 min at 17.000 xg and 4 °C after vigorously inverting the tubes for 30 sec. The supernatant was carefully removed and the RNA containing pellet was washed by adding 200 μl 99% EtOH and centrifuging for 5 min at 12.000 xg and 4 °C. The supernatant was completely removed and the pellet was air-dried for 3 min. The pellet was subsequently resuspended in 13 ml RNase-free ddH2O.
The bacterial variable regions 1 and 2 of 16S rRNA genes were amplified as described by Rausch et al. (2016). RNA-Seq libraries were created with the TruSeq Stranded mRNA Library Kit (Illumina, San Diego, USA).
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Description |
H01814
mRNA
|
| Data processing |
16S rRNA: After assembling the reads using SeqPrep, subsequent identification of chimera with ChimeraSlayer and manual verification, chimera were removed from the data set. The sequences were further analyzes using QIIME 1.9.0.
RNA-Seq: After quality filtering (fastq_illumina_filter, prinseq-lite, cutadapt), sequencing reads were mapped against the Drosophila melanogaster reference BDGP6 using TopHat v2.1.0 with sensitive option. The read counts per transcript were calculated with the Python script HTSeq v0.6.1p1 using the mode ‘intersection-strict’.
Genome_build: Drosophila melanogaster BDGP6
Supplementary_files_format_and_content: *_counts.tsv: Tab-delimited text files include read counts per gene for each sample.
Supplementary_files_format_and_content: otu_table_mc50_w_tax_wochimera_ctrl_50filtered.txt: Tab-delimited text file includes read counts per OTU for each sample.
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| |
|
| Submission date |
Jul 18, 2019 |
| Last update date |
Jul 05, 2021 |
| Contact name |
Daniela Esser |
| Organization name |
University Hospital Schleswig-Holstein
|
| Department |
Institute of Clinical Chemistry
|
| Lab |
Neuroimmunology
|
| Street address |
Arnold-Heller-Str. 3
|
| City |
Kiel |
| ZIP/Postal code |
24105 |
| Country |
Germany |
| |
|
| Platform ID |
GPL23323 |
| Series (1) |
| GSE134485 |
Recurrent application of dietary restriction reduces tumor load and increases lifespan in an intestinal cancer model |
|
| Relations |
| BioSample |
SAMN12309922 |
| SRA |
SRX6463924 |
