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| Status |
Public on Jul 29, 2023 |
| Title |
S2_G6_M_R1 |
| Sample type |
SRA |
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| Source name |
Whole body
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| Organism |
Drosophila melanogaster |
| Characteristics |
developmental stage: Adult
Sex: Male
selection treatment: short
selection generation: 6
population replicate: 2
sample replicate: 1
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| Treatment protocol |
We created four populations selected for long and short night sleep and two control populations in two stages. In the first stage, we created an outbred population (the Sleep Advanced Intercross Population, SAIP) using the five longest- and five shortest-sleeping lines of the Drosophila Genetic Reference Panel (DGRP) (Mackay et al., Nature, 2012, PMID 22318601; Huang et al., Genome Res, 2014, PMID 24714809). We crossed the following DGRP lines in a full diallel cross: DGRP_38, DGRP_235, DGRP_310, DGRP_313, DGRP_335, DGRP_338, DGRP_365, DGRP_379, DGRP_808, and DGRP_832. Two virgin males and two virgin females from each of the 100 F1 crosses were mixed together, and 10 males and 10 females were placed into 20 bottles. The progeny from the bottles were mixed for 21 generations, with 20 males and 20 females placed in each bottle for the subsequent generations. After generation 21, the bottles were maintained by bottle-to-bottle tipping. In the second stage, we established six populations at generation 47 of the outbred population by randomly choosing 25 males and 25 virgin females from the SAIP for each bottle. We assayed night sleep duration as well as other sleep and activity measures in 100 males and 100 females in each population. In two of the populations, the 25 longest-sleeping males and females were chosen from each bottle as parents to establish replicate long-sleeping populations. We followed a similar procedure to establish two replicate short-sleeper populations, choosing the 25 shortest-sleeping males and 25 shortest-sleeping females in each population as parents for the next generation. The remaining two populations served as replicated unselected controls; the parents for these populations were chosen by randomly selecting 25 males and 25 females from each of these populations. We repeated this procedure for 13 generations, and flies were not mixed across replicate populations. For generation 0 and generations 2-13, we randomly froze virgin male and virgin female flies from the sleep assays for RNA extraction. Ten males and ten females were frozen in two same-sex replicates from each of the six populations to generate 24 RNA sequence profiles per generation. All flies were frozen for RNA extraction at the same circadian time (12:00 pm).
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| Growth protocol |
All flies were kept in the following controlled environmental conditions. They were reared in an incubator on standard Drosophila food (http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/bloomfood.htm) at 25°C, 60% humidity, and a 12:12-hour light:dark cycle. Prior to making sleep measurements, we collected and maintained male and female virgins at 20 flies to a same-sex vial for four days to control for social exposure. We assayed night sleep duration as well as other sleep and activity measures using the Drosophila Activity Monitoring System (DAMS) (Trikinetics, Waltham, MA). Night and day sleep duration, average sleep bout length, and bout number as well as sleep latency and waking activity were computed from the DAMS activity data using an in-house C# program (R. Sean Barnes, personal communication).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted using Qiazol (Qiagen, Hilden, Germany), followed with phenol-chloroform extraction, isopropanol precipitation, and DNase digestion (Qiagen, Hilden, Germany). Qiagen RNeasy MinElute Cleanup kits (Qiagen, Hilden, Germany) were used to purify RNA according to the manufacturer’s instructions. Total RNA yield was measured using a Nanodrop 8000 (Thermo Scientific, Waltham, MA). We used Agilent Bioanalyzer RNA chips on the 2100 Bioanalyzer system (Agilent, Santa Clara, CA) according to the manufacturer instructions to visually inspect electrophoregrams for RNA degradation.
PolyA-selected stranded mRNA libraries were constructed from 1 µg total RNA using Illumina TruSeq Stranded mRNA Sample Prep Kits (Illumina, San Diego, CA). We followed the manufacturer’s protocol with the exception of the PCR amplification. The PCR amplification was performed for 10 cycles rather than 15 in order to reduce potential over-amplification. We applied unique barcode adapters to each library. Libraries were pooled for sequencing. To achieve a minimum of 38 million 126-base read pairs, the pooled libraries were sequenced on multiple lanes of an Illumina HiSeq2500 (Illumina, San Diego, CA) using version 4 chemistry.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Short population 2, Generation 6, Male, Replicate 1
STH_322
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| Data processing |
Base calls and quality were assessed using RTA version 1.18.64 and CASAVA 1.8.2.
We used fastqc to evaluate the quality of each sequence (Babraham Institute, Cambridge, UK).
We used STAR to align reads to the genome (Dobin et al., 2013, PMID: 23104886) with the default parameters except that minimum and maximum intron sizes were coded as 2 and 268,107 based on the Flybase FB2015_04 release 6 version 07 genome.
We used HTSeq to count only the uniquely mapped reads (Anders et al., 2015, PMID: 25260700).
Read counts were normalized using the method described by Love et al., 2014 (PMID: 25516281)
Assembly: FlyBase FB2015_04 release 6 version 07
Supplementary files format and content: There are two supplemental tables. The first, 'Raw_read_counts.csv', contains the raw read counts per gene as evaluated using HTSeq. The second file, 'Normalized_read_counts.csv', are the DESeq-normalized read counts for each gene. Both files are comma-separated values.
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| Submission date |
May 10, 2022 |
| Last update date |
Jul 29, 2023 |
| Contact name |
Susan T Harbison |
| E-mail(s) |
[email protected]
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| Phone |
301-435-8787
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| Organization name |
National Heart Lung and Blood Institute
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| Department |
Center for Systems Biology
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| Lab |
Laboratory of Systems Genetics
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| Street address |
10 Center Drive, Bldg. 10, Rm. 7D13
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL17275 |
| Series (1) |
| GSE202600 |
Title Multi-generational mRNA sequence data of pooled Drosophila melanogaster male and female flies from populations selected for long or short night sleep |
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| Relations |
| BioSample |
SAMN28181112 |
| SRA |
SRX15227699 |
| 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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