GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM7647070

Query DataSets for GSM7647070

Status

Public on Apr 17, 2024

Title

CTCF_wt_adult_C-CTCF_2

Sample type

SRA

Source name

whole organism

Organism

Drosophila melanogaster

Characteristics

development stage: adult
protein: dCTCF
antibody source: rabbit
strain: Oregon-R
genotype: dCTCFwt

Treatment protocol

Chromatin was prepared from two- to three-day-old adult flies. 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-CTCF C, 1:200) or G (anti-CP190, 1:200) Dynabeads (Invitrogen) mixed with 200 µL of PBST. Then antibodies-Dynabeads complexes and anti-HA Magnetic beads (Pierce) were washed and equilibrated in nuclear lysis buffer. 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.

Growth protocol

Drosophila strains were maintained on standard medium at 25°C and 50-60% humidity.

Extracted molecule

genomic DNA

Extraction protocol

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).
Libraries were prepared according to manufacturers recommendations with small modifications. In short, 1-10ng of purified, RNase treated, and reverse cross-linked genomic DNA was end-repaired and terminal adenosine residues were added using the NEBNext reagents. Custom-made indexed adapters were ligated, after which the material was size selected at ~200-600 bp with Ampure XP beads (Beckman Coulter). PCR amplification was performed using PE1.0 and PE2.0 primers (custom-made) for 12 cycles for Input samples and 14 to 15 cycles for IP-ed samples using the Q5 Hot Start HiFi PCR Master Mix (NEB). The PCR-amplified library was purified using Ampure XP beads and its quality was assessed on a Bioanalyzer 2100 system (Agilent). The libraries were sequenced on aNovaSeq 6000 (Illumina) in pair-end mode.

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)
Chip-seq coverage tracks (BedGraph) were obtained using deepTools (Ramírez et al. 2014) bamCoverage function with bin width 50 bp and RPKM normalization.
Assembly: dm6
Supplementary files format and content: Processed data was obtained by deeptools bamCoverage (bedGraph)

Submission date

Jul 19, 2023

Last update date

Apr 17, 2024

Contact name

Natalia Klimenko

E-mail(s)

lklimenko@genebiology.ru

Phone

9150884603

Organization name

Institute of Gene Biology (IGB) of the Russian Academy of Sciences