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| Status |
Public on Dec 12, 2023 |
| Title |
Control-KK- HS m6A-IP SYS rep1 |
| Sample type |
SRA |
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| Source name |
head
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| Organism |
Drosophila melanogaster |
| Characteristics |
ip antibody: SYS
heat shock condition: 30 minute heat shock
genotype: DaGal4>control
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| Treatment protocol |
male progeny were collected and anesthetized. Tissue was collected in basal conditions, immediately post Heat stress (HS), flies were placed into clear vials (20 flies per vial) and were HS for 30 minutes at 38.5°C.
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| Growth protocol |
DaGal4> Control RNAi (Vienna 60100), DaGal4> dTrmt10A RNAi(Vienna 100720), or DaGal4> UAS-dTrmt10A (This work/ DGRC Stock 1619899) Drosophila crosses set up in 25 degree incubator. Male progeny collected 1-2 days post eclosion for treatment and RNA extraction.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For m6A-IP seq flies were flash frozen and head tissue was collected into cold RNAse free tubes. Total RNA was extracted from 200 Drosophila Heads per replicate using Trizol/ chloroform extraction. PolyA+ mRNA was obtained using NEBNext Poly(A) mRNA Magnetic Isolation Module. PolyA+ RNA was fragmented using the NEB Next Magnesium Fragmentation Module (NEB, E6150S) for 4 minutes at 95 °C for a 250 ng sample of polyA+ RNA, and RNA was repurified using the Zymo RNA clean & concentrator -5 kit (Zymo, R1013). 10% of the fragmented polyA+ RNA was saved as an input control for sequencing. M6A -immunoprecipitation was done using the EpiMark N6-Methyladenosine Enrichment kit protocol with some minor alteration. 30ul of protein G magnetic beads (NEB, #S1430) were washed and resuspended in IP buffer (150mM NaCl, 10mM Tris-HCL, 0.1% NP-40). 4ul of synaptic systems antibody (SYS, 202003) was conjugated to protein G-magnetic beads (NEB, S1430S) for 2 hours at 4°C. Beads/ antibody were washed twice in IP buffer. ~1μg PolyA+ RNA was incubated with beads/antibody in IP buffer supplemented with 0.1% SUPERase-In RNase Inhibitor (Thermo Fisher; AM2696) for 2 hours at 4°C. After incubation, RNA/beads/antibody are washed twice in IP buffer, twice in low salt IP buffer (50mM NaCl, 10mM Tris-HCL, 0.1% NP-40), and twice in high salt IP buffer (500mM NaCl, 10mM Tris-HCL, 0.1% NP-40). RNA is eluted from beads with 25μl of RLT buffer twice and elution was pooled and concentrated using Zymo RNA clean and concentrator kit-5 (R1015). Libraries were made using SMARTer Stranded Total RNA-Seq Kit V2 without rRNA depletion(takarabio, 634411) for IPed and input RNA, and sequenced using illumina HiSeq with 40M paired end reads (2x150bp). Library preparation and sequencing was done by Admera Health. Three biological replicates per genotype and condition were done with Synaptic Systems m6A antibody.
Libraries were made using SMARTer Stranded Total RNA-Seq Kit V2 without rRNA depletion (takarabio, 634411) for IPed and input RNA, sequenced using illumina HiSeq with 40M paired end reads (2x150bp).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| Description |
m6A-IP-seq
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| Data processing |
Paired-end FASTQs were trimmed using TrimGalore
Trimmed FASTQs were mapped using STAR-2.7.3a
Mapped BAM files were filtered for primary mapped reads in properly matching pairs using samtools
Count tables were produced for RNA-seq using an in-house R script utilizing the GenomicRanges summarizeOverlaps function
Differential expression tables were produced by DESeq2 from RNA-seq count tables
m6A-IP-seq was processed with MeTPeak for peak calling and RADAR for differential peak calling.
Assembly: dm6
Supplementary files format and content: bigWig files for each condition were produced using deepTools (bamCoverage to produce bw files from BAM files with --normalizeUsing CPM)
Library strategy: m6A-seq
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| Submission date |
Apr 11, 2023 |
| Last update date |
Dec 12, 2023 |
| Contact name |
Alexandra Perlegos |
| E-mail(s) |
alexperlegos@gmail.com
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| Organization name |
University of Pennsylvania
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| Department |
Biology
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| Lab |
Bonini
|
| Street address |
231 Lynch Laboratories
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL23702 |
| Series (1) |
| GSE229430 |
The tRNA methyltransferase dTrmt10A impacts m6A levels and the stress response in the Drosophila brain |
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| Relations |
| SRA |
SRX19934677 |
| BioSample |
SAMN34146161 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7163437_kk.HS.sys.e11b1.m6A.bw |
5.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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