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Sample GSM7663568

Query DataSets for GSM7663568

Status

Public on Mar 20, 2024

Title

Msl3_MlewtF_adult_2

Sample type

SRA

Source name

whole organism

Organism

Drosophila melanogaster

Characteristics

antibody source: rabbit
strain: Dmel\M{3xP3-RFP.attP}ZH-86Fb
tissue: whole organism
genotype: MLEwt

Treatment protocol

Chromatin was prepared from two- to three-day-old adult males. A 1 g of adult flies was ground in a mortar in liquid nitrogen and resuspended in 20 mL of buffer A (15 mM HEPES-KOH, pH 7.6, 60 mM KCl, 15 mM NaCl, 13 mM EDTA, 0.1 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 0.5% NP-40, 0.5 mM DTT) supplemented with 0.5 mM PMSF and Calbiochem Cocktail V. The suspension was then homogenized subsequently in a Potter and Dounce homogenizer with tight pestle, filtered through 100 µm Nylon Cell Strainer (Miltenyi Biotec, United States), and cross-linked with 1% formaldehyde for 15 min at room temperature. Cross-linking was stopped by adding glycine to a final concentration of 125 mM. The nuclei were washed with three 10-mL portions of wash buffer (15 mM HEPES-KOH, pH 7.6, 60 mM KCl, 15 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 0.1% NP-40, protease inhibitors), one 5-mL portion of nuclear lysis basic buffer (15 mM HEPES, pH 7.6, 140 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 1% Triton X-100, 0.5 mM DTT, 0.1% sodium deoxycholate, protease inhibitors) and resuspended in 1 mL of nuclear lysis buffer (15 mM HEPES, pH 7.6, 140 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 1% Triton X-100, 0.5 mM DTT, 0.1% sodium deoxycholate, 0.5% SLS, 0.1% SDS, protease inhibitors). The suspension was sonicated in a Covaris ME220 focused-ultrasonicator (40 alternating 15-s ON and 45-s OFF intervals, peak power 75, duty % factor 25), and 50-µL aliquots were used to test extent of sonication and to measure DNA concentration. Debris was removed by centrifugation at 14 000 g, 4°C, for 10 min, and chromatin was pre-cleared with Protein A Dynabeads (Invitrogen). Corresponding antibodies were incubated for 1 hour at room temperature with 20 µL aliquots of Protein A (anti-Msl1, 1:100; anti-Msl2, 1:100; anti-Msl3, 1:500) Dynabeads (Invitrogen) mixed with 200 µL of PBST. Then antibodies-Dynabeads complexes were washed and equilibrated in nuclear lysis buffer.

Extracted molecule

genomic DNA

Extraction protocol

Chromatin samples containing 10–20 µg of DNA equivalent in 200 µL of nuclear lysis buffer (2 µL aliquots of such pre-cleared chromatin being stored as input material) were incubated overnight, at 4°C, with antibodies-Dynabeads complexes. After 3 rounds of washing with lysis buffer supplemented with 500 mM NaCl, and TE buffer (10 mM Tris-HCl, pH 8; 1 mM EDTA), the DNA was eluted with elution buffer (50 mM Tris-HCl, pH 8.0; 1 mM EDTA, 1% SDS), the cross-links were reversed, and the precipitated DNA was extracted by the ChIP DNA Clean &Concentrator kit (Zymoresearch).
The ChIP-seq libraries were prepared with NEBNext®_Ultra™_II DNA Library Prep kit, as described in the manufacturer's instructions. Amplified libraries were quantified using fluorometry with DS-11 (DeNovix, United States) and Bioanalyzer 2100 (Agilent, United States). Diluted libraries were clustered on a pair-read flowcell and sequenced using a NovaSeq 6000 system (Illumina, United States).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NovaSeq 6000

Data processing

Adapters, poly-N and poly-A read ends were removed using cutadapt software (Martin 2011). Cutadapt was also used for trimming low-quality ends (quality threshold was set to 20, reads with the length less than 20 bp after trimming were discarded).
The remaining reads were aligned to build version dm6 of the Drosophila melanogaster genome using Bowtie version 2 (Langmead and Salzberg 2012). Only reads that aligned concordantly exactly one time were passed to further analysis.
After alignment, read duplicates were removed using Picard MarkDuplicates function (http://broadinstitute.github.io/picard/). Also, peaks overlapping with blacklist regions were discarded (blacklist regions were previously converted from dm3 to dm6 genome built version) (https://sites.google.com/site/anshulkundaje/projects/blacklists)
Peak calling was performed using MACS version 2 against preimmune control (Zhang et al. 2008) in paired-end mode (option format=BAMPE).
Peaks with p value less than 1x10-2 were passed to the IDR pipeline to access reproducibility of chip-seq replicates (https://sites.google.com/site/anshulkundaje/projects/idr).
Optimal set of reproduced peaks was chosen for each sample and passed to further analysis.
Assembly: dm6
Supplementary files format and content: Processed data was obtained by MACS2 and deeptools bamCoverage (binSize 50) and includes: extended bed (narrowPeak) peak calls regions and coverage (bedGraph)

Submission date

Jul 26, 2023

Last update date

Mar 20, 2024

Contact name

Anastasia Revel-Muroz

E-mail(s)

revelanastasia@gmail.com

Organization name

Institute of Gene Biology, Russian Academy of Sciences

Department

Center for Precision Genome Editing and Genetic Technologies for Biomedicine