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Sample GSM4048302

Query DataSets for GSM4048302

Status

Public on Jan 01, 2020

Title

ZAF1_repl2

Sample type

SRA

Source name

whole organism

Organism

Drosophila melanogaster

Characteristics

strain: Oregon-R
genotype: WT
developmental stage: Embryonic
tissue: Whole body
chip antibody: rat anti-ZAF1 (aa 105-225)

Treatment protocol

Approximately 1g of Drosophila embryos were resuspended in 15ml ice-cold 0.1% Triton X-100 in PBS with protease inhibitors (PIs) and dounced 20x with loose pestle on ice, followed by centrifugation at 400g for 1min at 4 degrees. The supernatant was transferred to a fresh tube and centrifuged at 1100g for 10min at 4 degrees. The pellet was resuspended in 15ml ice-cold Cell lysis buffer with PIs (5 mM HEPES pH 8.0, 85 mM KCl, 0.5% NP40) and dounced 20x with tight pestle. Two equal aliquots were transferred into 15ml falcon tubes and centrifuged at 2000g for 4min at 4 degrees. The pellets consisting of nuclei were resuspended in 1ml of ice-cold Nuclear Lysis Buffer with PIs (50 mM HEPES pH 8.0, 10 mM EDTA, 0.5% N-laurylsarcosine) and incubated for 20min at RT. 1ml ice-cold Nuclear Lysis Buffer with PIs was added to each tube and sonicated using the bioruptor. Chromatin was then transferred to 1.5 ml-eppendorf tubes and centrifuged at 14000 rpm for 10 min at 4 degrs. Finally, supernatants were pooled, aliquoted and frozen in liquid nitrogen. Chromatin (50 ug of DNA) was brought to RIPA conditions (140mM NaCl / 10mM Tris-HCl pH8,0 / 1mM EDTA / 1% TritonX100 / 0,1% SDS / 0,1% sodium deoxycholate, 1mM PMSF) and incubated with anti-ZAF1, anti-dCTCF antibodies and rat IgG at 4 degrees on a rotary shaker overnight. The antibody/antigen complexes were precipitated with 40 ul protein G sepharose at 4 degrees for 3 hrs and washed 1x with RIPA buffer, 3x with RIPA buffer with 500 mM NaCl.

Growth protocol

Wild-type Drosophila melanogaster (Oregon R) flies were grown in population cages at 25 degrees. Embryos were collected for 12 hours. The collected embryos were dechorionated using 50% bleach and fixed in 10 ml cross-linking solution (50 mM HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 1.8% formaldehyde, pH 8.0) with 30ml n-heptane on a shaker table at RT for 15 minutes. The reaction was terminated with 125 mM glycine, 0.1% Triton X-100 in PBS. After washing out the fix, the embryos were blotted dry and frozen in liquid nitrogen. A small number of embryos from each collection were set aside for staging.

Extracted molecule

genomic DNA

Extraction protocol

Chromatin for input (0.5 ug of DNA) was brought to RIPA conditions (140mM NaCl / 10mM Tris-HCl pH8,0 / 1mM EDTA / 1% TritonX100 / 0,1% SDS / 0,1% sodium deoxycholate, 1mM PMSF) was diluted in TE with 0.5% SDS, treated with RNase A for 30 min and protease K overnight, incubated at 65 degrees for 6 hours and phenol chloroform extracted. Sepharose was resuspended in TE with 0.5% SDS, treated with RNase A for 30 min and protease K overnight, incubated at 65 degrees for 6 hours and phenol chloroform extracted.
Libraries were prepared according to manufacturer's 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 a HiSeq 2000 (Illumina) in single end mode.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

Read adapters and poly-A tails were removed using cutadapt software. Trimming of low-quality tails was performed using sickle in single-end mode, 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 (with enabling options --best, --strata, --tryhard). Only reads that aligned exactly one time were passed to further analysis. After alignment read duplicates were removed using Picard MarkDuplicates function.
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 with suppression of model building and assigning reads extension length to 200.
For samples with one biological replicate (dCTCF) peaks with p value less than 0.00001 were selected. For samples with two replicates (ZAF1) peaks with p value less than 0.1 were passed to IDR pipeline to access reproducibility of chip-seq replicates. As far as Rescue Ratio (RR) and Self-consistency Ratio (SR) passed recommended thresholds (RR and SR were less than 2) two merged replicates was chosen for further analysis (ZAF1_repl1 + ZAF1_repl2) with idr p value threshold 0.05.
Genome_build: dm6 (Release 6 plus ISO1 MT)
Supplementary_files_format_and_content: Processed data was obtained by MACS2 and includes: bedGraph (bedgraph) read pileups and extended bed (narrowPeak) peak calls regions.

Submission date

Aug 27, 2019

Last update date

Jan 01, 2020

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