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| Status |
Public on Oct 29, 2022 |
| Title |
Ad libitum male brain biol rep 2 |
| Sample type |
SRA |
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| Source name |
Brain
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| Organism |
Nothobranchius furzeri |
| Characteristics |
tissue: Brain
strain: GRZ
genotype: WT
treatment: AL - ad libitum
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| Treatment protocol |
For ad libitum treatment, fish were fed 7 feedings of 5 mg per feeding spread throughout from 7 am to 7 pm (i.e., feed 5 mg every 2 hours). For dietary restriction treatment, fish were fed 3 feedings of 5 mg per feeding in the morning (from 7 am to 9 am). Dietary treatment began when the fish was placed into 2.8 L tanks at 4 weeks of age and lasted for 5 weeks. Brains and livers were harvested from these killifish in two batches on two days (4 males and 4 females on each day). Fish were randomly assigned to a harvest batch, with each batch having 2 fish from each condition (AL-Male, DR-Male, AL-Female, DR-Female). On harvest day, the automatic feeders were first removed from the tank. Next, 1.5 h after the light turned on, for AL treatment, fish were manually fed 17.5 mg of dry food; and for DR treatment, fish were fed 7.5 mg of dry food. This quantity of food corresponded to half of the total daily food amount and maintained the differential ratio of food intake for the two diet regimens. From 12 PM to 3 PM, 8 fish were euthanized on ice, and organs were extracted and snap-frozen in liquid nitrogen. All organs were stored at -80˚C until RNA isolation.
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| Growth protocol |
All fish were raised from embryos collected from 15 male-female pairs, when these breeders were 9-10 weeks of age (within their reproductive peak). Collected embryos were treated with mild iodine (0.2%, diluted from Povidone-iodine solution (10% w/v, 1% w/v available iodine, RICCA #3955-16)) on Day 1 to reduce contamination, incubated in embryo solution with 0.01% methylene blue (Kordo, #37344), and then placed on moist coco fiber on the 14th day after collection. After two weeks on coco fiber, fish were hatched in ~1 cm-deep cold 1 g/L humic acid solution, at room temperature. For the next four days, the fish were housed on the countertop of the animal facility at 25C. System water was added to the hatching containers, and fish were fed live brine shrimp daily. For the following 2 weeks, fish were housed at a density of 4 fish per 0.8 L tank and fed with brine shrimps daily. In the following 2 weeks, housing density was reduced to 2 fish per 0.8 L tank and fish were fed as before. After 4 weeks post hatching, the fish was individually placed in 2.8 L tanks (singly housed) and fed dry food pellets according to specific dietary treatments.
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| Extracted molecule |
total RNA |
| Extraction protocol |
We isolated RNA from liver and brain using Qiagen RNeasy Mini kit (Qiagen, #74106) according the manufacturer’s instructions. The processing order of each organ was randomized. The tissues were transferred to the 1.2 mL Collection Microtubes (Qiagen, #19560) on dry ice in a 4°C cold room to reduce tissue thawing. Next, an autoclaved metal bead (Qiagen, #69997) and 700 µL of QIAzol (Qiagen, #79306) were added to each tube. Two rounds of tissue homogenization were performed on the TissueLyserII machine (Qiagen, #85300) at 25 Hz, 5 min each, at room temperature. The lysate was transferred to new 1.5 mL tubes, 140 µL chloroform (Fisher Scientific, #C298-500) was added, and the tubes were vortexed for 15 sec. After incubation at room temperature for 2-3 min, lysates were centrifuged at 12,000 x g at 4°C for 15 min. The aqueous phase was mixed with 350 µL ethanol (200 Proof, Gold Shield Distributors, #412804) by inverting the tubes 10 times. The mixture was transferred to the RNeasy column, washed with 350 µL RW1 buffer (provided by the RNeasy Mini kit), and treated with DNase I (following the kit’s protocol) at room temperature, for 15 min. The column was washed 2 times with 500 µL RPE buffers, and the RNA was eluted with 50 µL nuclease-free water (Invitrogen, #10977023). RNA quality and concentration were measured using an Agilent 2100 Bioanalyzer and the Agilent Nano Eukaryotic RNA Kit (Agilent, #5067-1511).
10 ng of RNA of each sample was used for cDNA synthesis. cDNA synthesis was performed using the Takara SMART-seq v4 PLUS kit (Takara, #634889) according to the manufacturer’s instructions. The cDNA libraries were prepared using the Illumina NexteraXT DNA library prep kit (Illumina, #FC-131-1096) based on the manufacturer’s protocol. The Illumina Nextera XT Index Kit v2 (Illumina, #131-2001) was used for library indexing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw sequencing data (FASTQ files) were processed and checked for quality using Trim-galore v0.4.5.
The processed reads were aligned to the African turquoise killifish reference genome downloaded from NCBI (Nfu_20140520, GCF_001465895.1). The read alignment was performed using STAR v2.7.1a with the default parameters.
Next, we used samtools v1.5 to remove the reads that map to multiple genomic regions, using the parameters of MAPQ<255 (“samtools view -q255 -b”).
These uniquely mapped reads were then used to generate the read counts for each gene, for which we used the “featureCounts” program from subread v2.0.1 using the default parameters.
We used DESeq2 v1.32.0 to analyze the differential gene expression.
Assembly: NCBI African turquoise killifish reference genome (Nfu_20140520, GCF_001465895.1).
Supplementary files format and content: Comma-separated text file includes raw counts for all samples in a matrix ("Counts_ALDR_220325.csv").
Supplementary files format and content: Comma-separated text file includes normalized counts for all liver samples in a matrix ("CountsNormDEseq2_Liver_ALDR_220331.csv").
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| Submission date |
Oct 22, 2022 |
| Last update date |
Oct 29, 2022 |
| Contact name |
Jingxun Chen |
| E-mail(s) |
[email protected]
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| Organization name |
Stanford University - Stanford, CA
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| Street address |
290 Jane Stanford Way, Room E365
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL32658 |
| Series (1) |
| GSE216369 |
An automated feeding system for the African killifish reveals effects of dietary restriction on lifespan and allows scalable assessment of associative learning |
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| Relations |
| BioSample |
SAMN31415744 |
| SRA |
SRX17995924 |
| 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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