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| Status |
Public on Feb 07, 2023 |
| Title |
Rhino Kdm3 GLKD sisters (control) biol rep 1 |
| Sample type |
SRA |
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| Source name |
ovary
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: ovary
cell line: na
cell type: germinal + somatic
genotype: wild type for Kdm3
treatment: none
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| Growth protocol |
Flies were raised at 25°C on cornmeal medium.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq: Chromatin immunoprecipitation (ChIP) was performed as described in Lee et al., 2006. Briefly, 100 ovary pairs were manually dissected into Schneider media and cross-linked in 1% formaldehyde/PBS for 10 min at room temperature with agitation. The cross-linking reaction was quenched by STOP buffer (PBS 1X, Triton 0.1%, Glycine 1 M) and ovaries were washed in PBS and homogenized in a glass douncer: first slightly dounced in PBST 0.1% and centrifugated 1 min 400g, followed by strong douncing in cell lysis buffer buffer (KCL 85 mM, HEPES 5 mM, NP-40 0.5% , Sodium butyrate 10 mM, EDTA free protease inhibitor cocktail Sigma) following by 5 min centrifugation at 2000g. We performed 2 washes with cell lysis buffer. The homogenates were then lysed on ice for 30 min in nucleus lysis buffer (HEPES 50 mM, EDTA 10 mM, N lauryl sarkosyl 0.5%, sodium butyrate 10 mM, EDTA free protease inhibitor cocktail Sigma). DNA was sheared using a Bioruptor pico from Diagenode for 10 cycles (30 sec on, 30 sec off). The sonicated lysates were cleared by centrifugation and then incubated overnight at 4°C with anti-Rhino antibody. Then 40 µL of Protein A Dynabeads was then added and allowed to bind antibody complexes by incubation for 1 h at 4°C. For H3K9me2 and me3 ChIP, 50 µL of Protein A Dynabeads were first coated with the anti-H3K9me3 or me2 antibodies and then incubated with the chromatin overnight at 4°C. Following four washing steps with high salt buffer (Tris pH 7.5 50 mM, NaCl 500 mM, Triton 0.25% , NP-40 0.5% , BSA 0.5% , EDTA pH 7.5 5 mM), DNA-protein complexes were eluted and de-cross-linked 10 h at 65°C. RNA and protein was digested by RNase A and Proteinase K treatments, respectively, before purification using Phenol/Chloroform protocol.
Barcoded libraries were prepared using Illumina technology
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
MACS2_narrowpeak_Rhino_Ctrl
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| Data processing |
small RNA-seq
Sequence reads in fastq format were trimmed from the adapter sequence 5’-TGGAATTCTCGGGTGCCAAG-3’ and matched to the D. melanogaster genome release 6 (dm6) using Bowtie
Sequence length distributions, small RNA mapping and small RNA overlap signatures were generated from bowtie alignments using Python and R (http://www.r- project.org/) scripts, which were wrapped and run in Galaxy instance publicly from ARTbio platform available at http://mississippi.fr.
For library comparisons, read counts were normalized to one million reads
unique mapper reads were obtained using sR_Bowtie -m0 (Galaxy Version 2.1.1)
For alignments we used a cleaned version of r6.36 version of Drosophila melanogaster genome (dm6, Flybase) in which sequences corresponding to unmapped (chrU), Y chromosome (chrY) and mitochondrial chromosome (chrM) were removed (dm6_clean).
Assembly: dm6_clean
Supplementary files format and content: bigWig, DEseq2 result for biological triplicates as a tab-delimited text file
RNA-seq
A DeSEq2 analyze on all D. mel. genes (release 6.36) was performed using the three biological replicates for each genotype, control or Kdm3 GLKD.
Assembly: dm6_clean
Supplementary files format and content: bigWig, DEseq2 result for biological triplicates as a tab-delimited text file
ChIP-Seq
reads were mapped on dm6_clean reference genome with BWA (Galaxy Version 0.7.17.4).
PCR duplicates were discarded using Picard tool (Galaxy Version 2.18.2.1)
Peak calling was made on mapped reads with MACS2 (Galaxy Version 2.1.1.20160309.6) using following parameters : --gsize ‘120000000’ --keep-dup ‘1’ --qvalue ‘0.05’ --nomodel --extsize ‘200’ --shift ‘0’. Narrowpeak was used for libraries obtained with Rhino antibody and broadpeak calling was used for libraries obtained with H3K9me2 and H3K9me3 antibodies ( --broad --broadcutoff=’0.1’).
Assembly: dm6_clean
Supplementary files format and content: bigWig and MACS2 files, broadpeak for H3K9me2 and H3K9me3, narrowpeak for Rhino
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| Submission date |
May 18, 2022 |
| Last update date |
Feb 08, 2023 |
| Contact name |
Antoine Boivin |
| E-mail(s) |
[email protected]
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| Organization name |
Sorbonne Universite CNRS
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| Department |
IBPS
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| Lab |
LBD UMR7622
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| Street address |
9 quai St Bernard
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| City |
PARIS |
| ZIP/Postal code |
75005 |
| Country |
France |
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| Platform ID |
GPL17275 |
| Series (1) |
| GSE203279 |
The histone demethylase KDM3 prevents auto-immune piRNAs production in Drosophila |
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| Relations |
| BioSample |
SAMN28525441 |
| SRA |
SRX15329086 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6165885_bamCompare_Rhino_Ctrl1.bigwig |
18.5 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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