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| Status |
Public on Jun 21, 2017 |
| Title |
213G |
| Sample type |
SRA |
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| Source name |
mushroom bodies
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| Organism |
Apis mellifera |
| Characteristics |
tissue: brain
colony: 6 (R23)
behavioural_type: Guard
n_reads: 49255298
mapped_apimel4.5: 40783416
mapped_dwv: 419953
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| Treatment protocol |
Groups of 10 individually marked seven-day-old adult bees were held in Petri dishes inside an incubator room (34° C ± 1° C, 50% ± 10% RH). The bees were held in the dish from adult emergence with ad libitum food supply and were not exposed to outside stimuli until the beginning of the experiment. All behavioural observations were performed in the incubator room, which mimics the hive environment. Observations were performed in white light. For defining bees’ behavioural types within a group, we developed a new assay using two behavioural subassays: the well-established resident-intruder assay8 and a the nursing assay 10. Briefly, the resident-intruder assay is a five minute survey of all aggressive interactions with an unrelated bee following its introduction to the groups; typically, about 3 bees will react with highly aggressive behaviours, attacking the intruder with biting and stinging. The nursing assay is a five minute survey of all nurturing interactions with a four-days-old queen larva in queen cup that is introduced to the groups; bees in the groups respond to the queen cell by entering the cell for inspection (short visits of less than 10 sec) or larval nursing (long visits of 11 sec or more). Typically, 2-3 bees will conduct nursing behaviour for more than 30s during the assay. Both subassays were done on the same group one after the other in random order according to a coin flip, then both subassays were repeated in random order an hour after the completion of the first set of subassays. Bees conducting highly aggressive behaviour including biting and stinging in both intruder assay were consider guard (G) bees; bees conducting larval nursing twice were consider nurses (N). Bees that didn’t respond with any overt social behaviour were defined as unresponsive (U) bees. The rest of the bees in the group were defined by their behaviour, which included “low responders”, bees that showed weak responses to a social stimulus, and animals that showed hybrid responses. Only the well-defined bees of the three behavioural types were used for further analysis. Individual observers conducted each of the four behavioural subassays in the social responsivity assay blind to any former observation while tracking the bees. The behavioural experiment was repeated with seven different colonies and the average of the percentage of bees in each behavioural type was calculated.
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| Growth protocol |
One-day-old adult bees were obtained by removing frames of honeycomb containing pupae from colonies, placing them in an incubator (34°C, 50 ± 5% relative humidity), and then monitoring their emergence every 24 h. One-day-old bees were individually marked on their thoraces with a spot of paint (Testor’s PLA) and placed in groups. Bees were kept in the laboratory in vertically oriented Petri dishes (100 X 20 mm) with a beeswax foundation sheet placed on the “wall” of the dish to mimic in-hive conditions. Dishes were supplied with one tube of honey (~1.4 mL), 30% sucrose solution (2 mL) and a mixture of fresh frozen pollen and 30% sugar solution (~10 mm diameter ball).
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| Extracted molecule |
total RNA |
| Extraction protocol |
The groups included in the gene expression study included well distinguished bees of each the three behavioural types: nurses, guards, and unresponsive bees. The bees were collected by flash freezing in liquid nitrogen and were placed in marked 1.5 mL microcentrifuge tube in a dry ice bucket. The bees were kept at -80° C until tissue collection. Six groups of bees from each of two SDI colonies were included in the gene expression analysis. Bees’ heads were separated from bodies on dry ice and placed in a dissection dish with 200 proof ethanol and dry ice to prevent RNA degradation during dissection. The cuticle of the head capsule was removed and each head was placed in RNAlaterICE (Life Technologies) at -20ºC for 14 h to 18 h. The heads were then opened, the hypopharyngeal glands removed, and the whole brain removed. Using a fine scalpel, the optic lobes were removed and a horizontal incision was made across the midbrain through the posterior protocerebral lobe. The lower part of the midbrain containing the antennal lobes and the subesophageal ganglion were removed. The upper part of the midbrain containing mostly the mushroom bodies (MB) and the central brain was used for gene expression analysis. MB were placed in a new 1.5 mL microcentrifuge tube and kept frozen at -80° C until RNA extraction. RNA extraction was performed with a PicoPure RNA Isolation Kit (Applied Biosystems; Lot #1210063) according to the manufacturer’s specifications, including a DNase treatment (QIAGEN) to remove genomic DNA contamination. 550 ng RNA from each sample were used for whole mRNA expression analysis.
The RNA-Seq libraries were constructed with the TruSeq® Stranded mRNA HT (Illumina) using an ePMotion 5075 robot (Eppendorf) according to manufacturer's specifications. The libraries were pooled in equimolar concentration as instructed and each pool was quantitated by qPCR and sequenced for 101 cycles on a HiSeq2500 using a HiSeq SBS sequencing kit v. 4.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
FASTQ files were generated with Casava v. 1.8.2
Sequence files were aligned to the Amel_4.5 genome (OGSv3.2 annotation build) using Tophat2 v. 2.0.10. with Bowtie2 v. 2.1.0 and Samtools v. 0.1.19.
Contamination by deformed wing virus (DWV) was calculated by aligning to the Pennsylvania variant of the DWV genome (GenBank accession: AY292384) using Bowtie 2 v. 2.1.0. Though there was some DWV contamination, the levels were an order of magnitude lower than experiments where DWV has been problematic.
Reads inside of gene features were counted using htseq-count in the HTSeq Python framework v. 0.6.1
Genome_build: Amel_4.5 (OGSv3.2 annotation) with Pennsylvania build of DWV (GenBank accession: AY292384)
Supplementary_files_format_and_content: Tab-delimited text files containing gene IDs and counts of reads mapped inside each gene feature.
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| Submission date |
Sep 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael C. Saul |
| E-mail(s) |
michael.saul@jax.org
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| Organization name |
The Jackson Laboratory
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| Lab |
Chesler Lab
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| Street address |
600 Main St
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| City |
Bar Harbor |
| State/province |
ME |
| ZIP/Postal code |
04609 |
| Country |
USA |
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| Platform ID |
GPL19174 |
| Series (1) |
| GSE87001 |
Deep evolutionary conservation of autism-related genes |
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| Relations |
| BioSample |
SAMN05781356 |
| SRA |
SRX2170153 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2318674_213G_CGCTCATT-TATAGCCT_L002_R1_001_gene_id.counts.txt.gz |
61.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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