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| Status |
Public on Oct 18, 2017 |
| Title |
dpse_TX_m_r4 |
| Sample type |
SRA |
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| Source name |
thorax without digestive system
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| Organism |
Drosophila pseudoobscura |
| Characteristics |
flybase species id: FBsp00000201
strain (strain name or flybase id or drosophila species stock center id): 14011-0121.94
developmental stage: adult
tissue: thorax without digestive system
Sex: Male
age post eclosion (days): 7-14
fly culture temperature (oc): 23
replicate: 4
illumina barcode: TCCGGAGA-CCTATCCT
plate and well id: Plate1_C2
ercc infomation: without ERCC
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| Growth protocol |
D. melanogaster stocks were obtained from the Drosophila Stock Center (Bloomington IN, NIH P40OD018537, http://fly.bio.indiana.edu), and were reared at the NIH (Bethesda MD). Fly food for D. melanogaster was a medium of water (8.5 L), agar (79 g), torumel yeast (275 g), cornmeal (520 g), granulated sugar (1,100 g), p-hydroxy-benzoic acid methyl ester (23.8 g), and 95% ethanol (91.8 mL) (LabExpress, Ann Arbor MI). All non-melanogaster flies were reared and dissected at the Drosophila Species Stock Center (San Diego CA, NSF CSBR 1351502, https://stockcenter.ucsd.edu). D. yakuba, D. ananassae, D. pseudoobscura, D. willistoni, and D. virilis were grown on a medium of de-ionized water (28 L), agar (300 g), soy flour (700 g), cornmeal (2,940 g), molasses (2,230 g), yeast (860 g), propionate (135 g), and nipogen (200 g) (https://stockcenter.ucsd.edu/info/food_cornmeal.php). D. persimilis and D. mojavensis were grown on a medium of agar (85 g), yeast (165 g), methylparaben (13.4 g), blended bananas (825 g), syrup (570 g), liquid malt extract (180 g), and 100% ethanol (134 mL) (https://stockcenter.ucsd.edu/info/food_banana_Opuntia.php). All flies were reared at 23-25 oC.
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| Extracted molecule |
polyA RNA |
| Extraction 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).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
mixed-species [D. melanogaster (w1118), D. pseudoobscura, and D. virilis] RNA preparation
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| Data processing |
De-multiplexed reads (Chastity > 0.6) were generated by Illumina CASAVA (v.1.8.2) as fastq files. For the single-species samples (i.e., D. melanogaster genitalia samples), we provided the raw de-multiplexed fastq files without processing. For the multi-species samples, we first made merged genome and annotation of appropriate Drosophila species plus corrected ERCC standards (Lee et al., Journal of Genomics, 2016, PMID 27512518). Additionally, we added a four-letter prefix for each chromosome or scaffold in the merged genome or annotation (e.g., 2L => dmel_2L). ERCC names were unchanged due to their specific prefix. The de-multiplexed reads from the multi-species samples were then mapped to the appropriate merged genome and annotation using STAR (v2.4.2a) (Doblin et al., Bioinformatics, 2012, PMID 23104886) with default parameters. We used an in-house python script to fetch the prefix of read names in the bam alignments, and obtained all uniquely-mapped and multi-hit (2-10 hits) reads (in bam format) that were mapped exclusively to a single species or exclusively to ERCC. We converted the bam reads to fastq reads by BEDTools (2.25.0; bamtofastq) (Quinlan and Hall, 2010, Bioinformatics, PMID 20110278). After this step, reads of each multi-species sample were split into five categories – species 1, species 2, species 3, ERCC, and leftover, which includes unmapped reads and reads mapped to multiple species (or multiple species plus ERCC). For multi-species samples, the original fastq file can be restored by merging the reads of appropriate three species as well as the ERCC and leftover reads.
We provide both STAR (SRR5639* and SRR6181*) and HiSAT2 (SRR724*) fastq and associated matrices of raw gene-level read counts in this GEO entry. For STAR read count files, we used the FlyBase (FB) annotations (version 2017_03). For HiSAT2 read count files, we used both FB and updated (YO) annotations (data in Supplementary Files of this GEO). For the latter one, we also provide two extra columns in the expression matrix, showing the maximum exon-level Jaccard index overlap between our YO annotation gene IDs and FlyBase ones. In addition, we provided bigWig tracks for all samples (replicated merged), YO annotation files (in both gtf and gff3 format), ortholog look up table (among YO gene ID, FlyBase gene ID, and dmel gene), gene-level normalized read counts based on either YO or FlyBase annotation, and transcript-level transcripts per million (TPM) based on YO or FlyBase annotation.
Supplementary_files_format_and_content: counts
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| Submission date |
Oct 17, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Brian Oliver |
| E-mail(s) |
briano@nih.gov
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| Phone |
301-204-9463
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| Organization name |
NIDDK, NIH
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| Department |
LBG
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| Lab |
Developmental Genomics
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| Street address |
50 South Drive
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL13310 |
| Series (1) |
| GSE99574 |
RNA-seq of sexed adult tissues/body parts from eight Drosophila species |
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| Relations |
| BioSample |
SAMN07795119 |
| SRA |
SRX3291675 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2817266_dpse_TX_m_r4.htseq_reverse.HiSAT2.FB.txt.gz |
61.6 Kb |
(ftp)(http) |
TXT |
| GSM2817266_dpse_TX_m_r4.htseq_reverse.HiSAT2.YO.txt.gz |
174.2 Kb |
(ftp)(http) |
TXT |
| GSM2817266_dpse_TX_m_r4.htseq_reverse.txt.gz |
59.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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