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SRX2877729: GSM2647020: dmoj_HD_f_r1; Drosophila mojavensis; RNA-Seq
2 ILLUMINA (Illumina HiSeq 2000) runs: 11.7M spots, 889.5M bases, 567.2Mb downloads

Submitted by: NCBI (GEO)
Study: RNA-seq of sexed adult tissues/body parts from eight Drosophila species
show Abstracthide Abstract
This data set contains polyA+ transcriptional profiling of sexed adult tissues/body parts and whole adults of eight Drosophila species: Drosophila melanogaster (FBsp00000001) from two strains [w1118 (FBst0005905) and Oregon-R (FBst0025211)], Drosophila yakuba (FBsp00000254), Drosophila ananassae (FBsp00000052), Drosophila pseudoobscura (FBsp00000201), Drosophila persimilis (FBsp00000188), Drosophila willistoni (FBsp00000253), Drosophila mojavensis (FBsp00000160), and Drosophila virilis (FBsp00000251). It is worth noting that the Samples (856) section of this series page only lists the top 500 samples, and the rest of the 356 samples are not shown in the list but are available in GEO by putting the GSM ID in the search box at the top of the page. All raw data are available by clicking the SRP108530 link (SRA section). It is noteworthy that all of the fourth replicate of our samples are included in the last 356 samples in this series. Please see the supplementary file "All_samples_with_title.txt" for a complete list of GSM IDs and sample titles. Overall design: Samples were collected in quadruplicate from sexually-mature freely-mated adults (4 to 14 days post-eclosion). Samples were either sexed whole flies (individual) or tissues/body parts (pooled from 2-5 flies). For the two strains of D. melanogaster, we profiled whole flies along with seven adult tissues/body parts for each sex. Dissection examples are shown in Figure 1. Samples were whole organism (FBbt:00003004), head (FBbt:00003007, Fig. 1A), thorax (FBbt:00003018, Fig. 1B) without digestive system, abdomen (FBbt:00003023, Fig. 1C) without digestive or reproductive system, gonad [female (FBbt:00004865, Fig. 1D) or male (FBbt:00004928, Fig. 1E)], reproductive system [female (FBbt:00004864, Fig. 1F) or male (FBbt:00004927, Fig. 1G)] without gonad or genitalia, genitalia [female (FBbt:00004827, Fig. 1H) or male (FBbt:00004828, Fig. 1I)], and digestive (FBbt:00003123) plus excretory (FBbt:00005724) system (Fig. 1J). For non-melanogaster species, we dissected as above except genitalia were included in the reproductive system without gonad. Non-melanogaster strains were: D. yakuba (Drosophila Species Stock Center ID: 14021-0261.01), D. ananassae (14021-0371.13), D. pseudoobscura (14011-0121.94), D. willistoni (14030-0811.24), D. virilis (15010-1051.87), D. persimilis (14011-0111.49), and D. mojavensis (15081-1352.22). After extracting RNA from each sample, we mixed equal amounts of total RNA (except genitalia samples from D. melanogaster) from each of three genetically distant species (rate of cross-mapping in simulations < 0.6%). Specifically, we mixed RNA of: 1) D. melanogaster (w1118), D. pseudoobscura, and D. virilis; 2) D. melanogaster (Oregon-R), D. mojavensis, and D. persimilis; 3) D. yakuba, D. ananassae, and D. willistoni. We isolated polyA+ RNA from these pools and prepared RNA-seq libraries, which were further multiplexed with indexed adaptors and pooled for sequencing.
Sample: dmoj_HD_f_r1
SAMN07187947 • SRS2249504 • All experiments • All runs
Library:
Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Flies were anesthetized using CO2, and all samples were dissected in phosphate buffered saline solution (PH 7.4, Life Technologies, Carlsbad CA) and transferred to 56 or 112 μL of RNAlater (Life Technologies, Carlsbad CA) in 96-well 1.5 mL plates at room temperature after being macerated with forceps. Plates were stored at -80 oC at the NIH after dissection (melanogaster samples) or after dissection and shipping (non-melanogaster samples).  For RNA extraction, we added 20-30 1.0 mm glass beads (BioSpec Products, Bartlesville OK) to each sample and added RLT buffer from the RNeasy 96 kit (Qiagen, Valencia CA) to 712 μL (final volume).  We homogenized the samples three times for 60 seconds each with a mini-beadbeater (Biospec Products, Bartlesville OK). Extraction of total RNA was performed using the RNeasy 96 kit (spin version), except that we used 712 μL of 70% ethanol due to the increased volumes of RNAlater/RLT mixture.  RNA was quantified using Quant-iT RiboGreen (Life Technologies, Carlsbad CA).  We spot checked RNA integrity by capillary electrophoresis on a TapeStation 2200 (Agilent Technologies, Santa Clara CA) and found that the rRNA bands were distinct. We used 33 ng aliquots of RNA of three genetically distant species for each library, except for thorax, where we used 17 ng due to lower yield. The mixing of RNA was performed using a pipetting robot (Andrew Alliance, Vernier CH). RNA from D. melanogaster genitalia was not mixed with tissues of the other species. We used 100 ng of total RNA as input (except thorax samples: 50 ng), and followed the TruSeq PolyA+ stranded mRNA kit protocol with half volume reactions (Illumina, San Diego CA).  For each sample, we also added 10 pg Standard Reference Material 2374 beta (Pool 78A or 78B) ERCC spike-ins (Lee et al., Journal of Genomics, 2016, PMID 27512518) to the Fragment-Prime-Finish Mix during the mRNA fragmentation step.  We used 96-plex molecular barcoding (Kozarewa and Turner, Methods in Molecular Biology, 2011, PMID 21431778) from the Illumina TruSeq kit for all samples. Libraries were quantified with Quant-iT PicoGreen (Life Technologies, Carlsbad CA). For each sample, we conducted RNA-seq for the three libraries with the highest DNA concentration out of four libraries. The quality (i.e., no overt primer dimers or low complexity) of all selected libraries were confirmed by capillary electrophoresis on a TapeStation 2200 (Agilent Technologies, Santa Clara CA). We performed single-end 76 bp PolyA+ sequencing on the HiSeq2000 Sequencing System (Illumina, San Diego CA).
Experiment attributes:
GEO Accession: GSM2647020
Links:
Runs: 2 runs, 11.7M spots, 889.5M bases, 567.2Mb
Run# of Spots# of BasesSizePublished
SRR56393295,897,402448.2M286.1Mb2017-06-05
SRR72432505,806,235441.3M281.1Mb2018-05-31

ID:
4119520

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