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| Status |
Public on Jan 18, 2019 |
| Title |
Dh44_2_mmPCR |
| Sample type |
SRA |
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| Source name |
diuretic hormone 44 neuron nuclei
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| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: UAS-unc84-2XGFP; Dh44-GAL4
neuronal population: diuretic hormone 44
replicate: 2
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| Growth protocol |
Flies were raised at 25°C in a 12-h light/12-h dark cycle in 60% relative humidity and maintaned on cornmeal, yeast, molasses, and agar medium. Approximately 300 heads were collected from 2-3 day old flies.
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
Heads were separated by vigorous vertexing followed by separation over dry-ice cooled sieves. 9ml of homogenization buffer (20mM β-Glycerophosphate pH7, 200mM NaCl, 2mM EDTA, 0.5% NP40 supplemented with RNAase inhibitor,10mg/ml t-RNA, 50mg/ml ultrapure BSA, 0.5mM Spermidine, 0.15mM Spermine and 140ul of carboxyl Dynabeads -270 Invitrogene: 14305D) was added to each sample. The heads were minced on ice by a series of mechanical grinding steps followed by filtering the homogenate using a 10um Partek filter assembly (Partek: 0400422314). After removing the carboxyl-coated Dynabeads using a magnet, the homogenate was filtered using a 1um pluriSelect filter (pluriSelect: 435000103). The liquid phase was carefully placed on a 40% optiprep cushion layer and centrifuged in a 4oC centrifuge for 30min at ~2300Xg. The homogenate/Optiprep interface was incubated with anti-GFP antibody (Invitrogen: G10362) and protein G Dynabeads (Invitrogen: 100-03D) for 40 minutes at 4oC . Beads were then washed once in NUN buffer (20mM β-Glycerophosphate pH7, 300mM NaCl, 1M Urea, 0.5% NP40, 2mM EDTA, 0.5mM Spermidine, 0.15mM Spermine, 1mM DTT, 1X Complete protease inhibitor, 0.075mg/ml Yeast torula RNA, 0.05Units/ul Superasin). Bead-bound nuclei were separated using a magnet stand, and RNA was extracted using the Picopure Kit (Invitrogen KIT0204).
We performed mmPCR-seq to quantify editing levels at 605 loci harboring know editing sites. We prepared samples for microfluidic PCR at 605 loci with a 15-cycle pre-amplification PCR reaction using 10 ul of cDNA made from INTACT RNA extractions, using the High capacity cDNA Reverse Transcriptase Kit, 6 ul of a pool of all primers used in the multiplex microfluidic PCR, and 4 ul of 5X KAPA2G Fast Multiplex. The pre-amplification reactions were purified using AMPure XP PCR purification beads. We loaded the pre-amplified samples and 48 pools of PCR primers into a 48.48 Access Array IFC (Fluidigm) and performed target amplification. Multiplex PCR products were barcoded using a 13-cycle PCR reaction.
microfluidic multiplex PCR and sequencing (mmPCR-seq)
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Heads were collected from a mixed population of males and females.
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| Data processing |
Low quality bases were trimmed from RNA-seq reads using Trim Galore and mapped to the dm6 using STAR (v2.4.2) --twopassMode.
The samtools mpileup function was used to determine base calls at known editing sites from uniquely mapped reads. Editing levels were calculated as (G reads)/(A+G reads) at each site.
Genome_build: dm6
Supplementary_files_format_and_content: A and G counts and editing levels at known editing sites. Tab-delimited files are formatted as: chr, position, a counts, g counts, editing level.
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| Submission date |
Apr 25, 2018 |
| Last update date |
Jan 20, 2019 |
| Contact name |
Galit Shohat-Ophir |
| E-mail(s) |
[email protected]
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| Organization name |
Bar Ilan University
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| Street address |
The Nanotechnology Building 206, Room C-663
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| City |
Ramat Gan |
| ZIP/Postal code |
5290002 |
| Country |
Israel |
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| Platform ID |
GPL19132 |
| Series (2) |
| GSE113662 |
Measuring A-to-I RNA editing signatures of neuronal populations within the Drosophila brain |
| GSE113663 |
A-to-I RNA editing signatures of neuronal populations within the Drosophila brain |
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| Relations |
| BioSample |
SAMN08981543 |
| SRA |
SRX3994128 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3110806_dh44_2_mmPCR_editing.txt.gz |
11.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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