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| Status |
Public on Jul 15, 2021 |
| Title |
PI_mouse |
| Sample type |
SRA |
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| Source name |
whole organism
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| Organism |
Drosophila melanogaster |
| Characteristics |
antibody source: mouse
strain: Oregon-R
genotype: wt
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| Treatment protocol |
Approximately 2g of Drosophila adult flies 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 Covaris. 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-HA, anti-dCTCF, anti-CP190 antibodies, rat and mouse IgG at 4 degrees on a rotary shaker overnight. The antibody/antigen complexes were precipitated with 20 ul protein G Dynabeads at 4 degrees for 3 hrs and washed 1x with RIPA buffer, 3x with RIPA buffer with 500 mM NaCl.
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| Growth protocol |
Wild-type Drosophila melanogaster (Oregon R) flies were grown in population cages at 25 degrees. 2 g of 2-3 day adult flies were frozen in liquid nitrogen.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Dynabeads were 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 extracted on columns (ChIP 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 a HiSeq 2000 (Illumina) in single end mode.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| 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 (IDR p value was set to 0.05 for true replicates and 0.01 for pseudoreplicates) was chosen for each sample and passed to further analysis.
Chip-seq coverage tracks (BedGraph) were obtained using deepTools (RamÃrez et al. 2014) bamCoverage function with bin width 100 bp and RPKM normalization.
Genome_build: dm6
Supplementary_files_format_and_content: Processed data was obtained by IDR and deeptools bamCoverage and includes: extended bed (narrowPeak) peak calls regions and coverage (bedGraph)
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| Submission date |
May 24, 2021 |
| Last update date |
Jul 15, 2021 |
| Contact name |
Natalia Klimenko |
| E-mail(s) |
lklimenko@genebiology.ru
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| Phone |
9150884603
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| Organization name |
Institute of Gene Biology (IGB) of the Russian Academy of Sciences
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| Street address |
34/5 Vavilova Street
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| City |
Moscow |
| ZIP/Postal code |
143026 |
| Country |
Russia |
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| Platform ID |
GPL25244 |
| Series (1) |
| GSE175402 |
Drosophila architectural protein CTCF is not essential for fly survival and is able to function independently of CP190 |
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| Relations |
| BioSample |
SAMN19316618 |
| SRA |
SRX10971119 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5333001_PI_mouse_100_RPKM.bedGraph.gz |
6.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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