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Sample GSM6165868

Query DataSets for GSM6165868

Status

Public on Feb 07, 2023

Title

Input Kdm3 GLKD, 1

Sample type

SRA

Source name

ovary

Organism

Drosophila melanogaster

Characteristics

tissue: ovary
cell line: na
cell type: germinal + somatic
genotype: Kdm3 germline knock down
treatment: none

Growth protocol

Flies were raised at 25°C on cornmeal medium.

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

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

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

Submission date

May 18, 2022

Last update date

Feb 10, 2023

Contact name

Antoine Boivin

E-mail(s)

[email protected]

Organization name

Sorbonne Universite CNRS

Department

IBPS