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| Status |
Public on May 08, 2019 |
| Title |
Zn72D-GFP_RIP_2 |
| Sample type |
SRA |
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| Source name |
Zn72D-GFP_RIP
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| Organism |
Drosophila melanogaster |
| Characteristics |
Sex: pooled male and female
developmental stage: adult
genotype/variation: y1 w*; P{PTT-GA}Zn72D[CA07703]
tissue: head
replicate: 2
molecule subtype: rRNA-depleted anti-GFP immunoprecipitated 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.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA immunoprecipitation was performed after homogenizing ~500 ul of fly heads in IP Buffer (150 mM NaCl, 20 mM HEPES pH 7.5, 2 mM MgCl2, 0.1% NP40, cOmplete protease inhibitor, RNaseOUT RNase inhibitor 1U/ul (Invitrogen)). Lysates were split in half, and 4% of input was removed for input control libraries. IP lysates were incubated overnight at 4C with Dynabeads Protein G (Invitrogen: 10003D), plus 5 ug of anti-GFP antibody (Abcam: ab290). IPs were washed 8 times in IP buffer. Beads with protein bound and saved inputs were added to 1 mL of TRIzol (Thermo Fisher: 15596026). 200 ul of chloroform was added, and samples were centrifuged at 14000 xg at 4C for 15 minutes. Aqueous phases were collected, mixed with 1 volume of 70% ethanol and then transferred to a RNeasy MinElute column (Qiagen, Hilden, Germany: 74204) for purification following the standard protocol.
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 HyperPrep RNA-seq Kits (Kapa Biosystems: KK8540) to make RIP-seq libraries for fly samples.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| 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 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 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) |
| GSE126630 |
Identifying RNAs bound by Drosophila Zn72D |
| GSE126631 |
Zinc finger RNA binding protein Zn72D regulates ADAR-mediated RNA editing in neurons |
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| Relations |
| BioSample |
SAMN10953766 |
| SRA |
SRX5383040 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3610105_Zn72D-GFP_RIP_2.genes.results.txt.gz |
903.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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