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| Status |
Public on May 08, 2019 |
| Title |
Dmel_Irp-1A_RNAi_1 |
| Sample type |
SRA |
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| Source name |
whole brain_Dmel_Irp-1A_RNAi
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| Organism |
Drosophila melanogaster |
| Characteristics |
Sex: female
genotype: C155-Gal4; UAS-Irp-1A_RNAi
age: 3-5 day old
tissue: whole brain
replicate: 1
molecule subtype: rRNA-depleted RNA
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| Growth protocol |
Flies were raised at 25C in a 12 hour light/dark cycle on molasses, cornmeal, agar, and yeast media. Brains of 3-5 day old adult females were dissected for RNA extractions.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from ~15 brains using RNAdvanced Tissue Kit (Beckman Coulter) followed by TURBO DNase treatment (Thermo Fisher).
150-250 ng of total RNA was mixed with an equal amount of pooled DNA oligos designed antisense to Drosophila rRNAs in 50 bp sections in an 8 ul reaction with 2ul of 5X Hybridization buffer (500 mM Tris-HCl pH 7.4, 1 M NaCl). rRNA antisense oligos were annealed to total RNA samples for 2 minutes at 95°C, the temperature was slowly reduced to 65°C and 2U of Thermostable Hybridase H (Epicentre) and 1 ul of 10X Digestion buffer (500 mM Tris-HCl, 1 M NaCl, 200mM MgCl2) were added to make 10 ul total and reactions were incubated for 30 minutes at 65°C. rRNA-depleted RNA was then purified using Agencourt RNAClean XP beads, treated with TURBO DNase (Ambion) and then purified with RNAClean XP beads again. rRNA-depleted RNA was used as input to the KAPA Stranded RNA-seq Kit (KK8400) to make RNA-sequencing libraries.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Paired-end, 76-base-pair reads were trimmed using Trim Galore 0.4.5 to remove adapter sequences.
Trimmed reads were mapped to the dm6 genome using STAR 2.5.4b with the parameters --outFilterScoreMin 10 --alignEndsType EndToEnd.
To call editing levels, samtools (v1.4) mpileup function was used to extract base calls from uniquely mapped reads at known editing sites. Editing levels were detemined as the number of G reads out of all A and G reads at a site.
To determine gene expression levels, RSEM 1.2.30 was used to count the number of reads mapping to each gene. RSEM calculated expected counts were rounded down to the nearest integer and used as input to DESeq2 v1.20.0
Genome_build: dm6
Supplementary_files_format_and_content: For RNA editing analysis: A and G counts and editing levels at known editing sites. Tab-delimited filtes are formatted as: chr, pos, A counts, G counts, editing level.
Supplementary_files_format_and_content: For expression analysis: genes.results output files from RSEM. Tab-delimited files are formatted as: gene_id, transcript_id(s), length, effective_length, expected_count, TPM, FPKM, posterior_mean_count, posterior_standard_deviation_of_count, pme_TPM, pme_FPKM, TPM_ci_lower_bound, TPM_ci_upper_bound, FPKM_ci_lower_bound, FPKM_ci_upper_bound.
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| Submission date |
Feb 15, 2019 |
| Last update date |
May 08, 2019 |
| Contact name |
Jin Billy Li |
| E-mail(s) |
[email protected]
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| Organization name |
Stanford University
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| Department |
Genetics
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| Street address |
300 Pasteur Dr, Alway M341
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL19132 |
| Series (2) |
| GSE126628 |
Measuring the influence of RNA binding proteins on A-to-I RNA editing in the Drosophila brain |
| GSE126631 |
Zinc finger RNA binding protein Zn72D regulates ADAR-mediated RNA editing in neurons |
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| Relations |
| BioSample |
SAMN10953697 |
| SRA |
SRX5382984 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3610065_Dmel_Irp1A_RNAi_1.genes.results.txt.gz |
864.2 Kb |
(ftp)(http) |
TXT |
| GSM3610065_Dmel_Irp1A_RNAi_1_editing.txt.gz |
83.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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