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Sample GSM5402009

Query DataSets for GSM5402009

Status

Public on Aug 26, 2022

Title

control-RNAi HS input NEB rep2

Sample type

SRA

Source name

head

Organism

Drosophila melanogaster

Characteristics

ip antibody: none
heat shock condition: 30 minute heat shock
genotype: control RNAi
tissue: head

Treatment protocol

Control and Mettl3 RNAi male progeny were collected and anesthetized. Tissue was collected in basal conditions, immediately post Heat stress (HS), or HS and recovered for 6hrs or 24hrs. For HS, flies were placed into clear vials (20 flies per vial) and were HS for 30 minutes at 38.5°C. For HS recovery, flies were HS for 30 minutes at 38.5°C and allowed to recover on regular food in the 25°C incubator for 6hr or 24 hrs.

Growth protocol

DaGal4> Control RNAi (BL35785) and DaGal4> Mettl3 RNAi(BL80448) Drosophila crosses set up in 25 degree incubator. Male progeny collected 1-2 days post eclosion for treatment and RNA extraction.

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). 1.4ul of NEB m6A antibody (NEB, E1610S), or 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 NEB m6A antibody, and two biological replicates were done with Synaptic Systems m6A antibody. Total RNA was extracted from brains using trizol/chloroform and Zymo RNA clean and concentrator kit-5 (R1015). The RNA-seq libraries from brains were prepared using the Tru-seq stranded mRNA library prep. Library preparation and sequencing was done by Admera Health, and sequenced using illumina HiSeq with 40M paired end reads (2x150bp). Three biological replicates were done for each experimental timepoint, condition, and genotype. Technical repeats and repeat additional biological replicates were done as indicated.
Libraries were made using SMARTer Stranded Total RNA-Seq Kit V2 without rRNA depletion (takarabio, 634411) for IPed and input RNA, The RNA-seq libraries from brains were prepared using the Tru-seq stranded mRNA library prep. Both m6A-IP and RNa-seq were sequenced using illumina HiSeq with 40M paired end reads (2x150bp).

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

HiSeq X Ten

Description

input for m6A-IP-seq
control_RNAi.HS.neb.log2_m6A_input.bw
read_counts.txt.gz

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.
Genome_build: dm6
Supplementary_files_format_and_content: bigWig files for each condition represent log2(normalized m6A-IP counts/normalized input counts) and were produced using deepTools (bamCoverage to produce bw files from BAM files with --normalizeUsing CPM, and bwCompare using --operation log2

Submission date

Jun 26, 2021

Last update date

Aug 26, 2022

Contact name

Emily Shields

E-mail(s)

emily@bonasiolab.org

Organization name

University of Pennsylvania

Street address

3400 Civic Center Blvd