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| Status |
Public on Feb 07, 2022 |
| Title |
BGBEAF-32- RNA-seq replicate 2 |
| Sample type |
SRA |
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| Source name |
BGBEAF-32- RNA-seq
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| Organism |
Drosophila melanogaster |
| Characteristics |
genotype/variation: BGBEAF-32-
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| Treatment protocol |
Primer sequences for CP190, Chro, Dref and BEAF-32 dsRNAi were obtained from the Drosophila RNAi Screening Center database (http://flyrnai.org) (see Table S5). The primers with T7 promoter sequence were used to amplify the IVT templates from wild type genomic DNA using Dream Taq DNA Polymerase Kit (Thermo Scientific, EP0703). The PCR products were checked by electrophoresis and purified using a Fastgene PCR Purification Kit (Fastgene). The purified PCR products were then used as templates to synthesis dsRNA using the MEGAscript T7 Kit (Life Technologies, AM1334) according to the manufacturer’s recommendations. The BG3 cells were transfected with 50ug of dsRNA using Fugene (Promega) according to manufacturer’s protocol. Cells were harvested after 72 hours and processed for downstream experiments accordingly.
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| Growth protocol |
BG3 cells were grown to 80-90% confluence in Schneider’s Drosophila medium with 10% FBS respectively and antibiotics (Pen-Strep) in 100 mm cell culture plates.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Hi-C libraries were generated from 10 million cells by following the Insitu Hi-C protocol as mentioned in (Rao et al. 2014) with minor modifications. Crosslinked cells were lysed and genome was digested using DpnII (NEB) overnight. The overhangs were filled with Bioton-16-dATP (Jena Bioscience) followed by ligation and de-crosslinking with proteinase K digestion. The sample was further sonicated using Bioruptor.
Biotinylated DNA was pulled down using Dynabeads MyOne Streptavidin T1 beads (Life technologies, 65602). Selected biotinylated DNA fragments ranging from 200-500bp were then ligated with illumina adaptors (NEB).
RNA extraction: RNA extraction was carried out using Trizol according to manufacturer’s instructions. RNA was further DNase treated and purified using RNeasy Mini kit (Qiagen) following manufacturer's protocol. RNA was assessed qualitatively and quantitatively using Quibit and Bioanalyzer 2100(Agilent).
RNA sequencing: PolyA RNA selection, library preparation and sequencing were carried out by Novogene.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
HiSeq Control Software HD 3.4.0.38 used for basecalling.
Hi-C analysis: Each pair of the PE reads were aligned separately to Drosophila melanogaster (dm6) genome (Adams et al., 2000; dos Santos et al., 2015) using BWA-mem (Li and Durbin, 2010) (with options -t 20 -A1 -B4 -E50 -L0). HiCExplorer was used to build and correct the contact matrices and detect TADs and enriched contacts (Ramirez et al., 2018). The contact matrices were built at 100 Kb bins for plotting Figures 1, S5 and S6 only, 10Kb for compartments (Rowley et al., 2017) and using the DpnII restriction sites for everything else. Using a minimum allowed distance between restriction sites of 150 bp and a maximum distance of 1000 bp, we obtained a matrix with 217638 bins with a median width of 529 bp. After filtering, we obtained between 18 M and 65 M reads (see Table S1). The matrices were corrected using the thresholds in Table S2, where values were selected from the diagnostic plots (Figure S8). By using the corrected contact matrices, we detected TADs of at least 5 Kb width using a P-value threshold of 0.01, a minimum threshold of the difference between the TAD-separation score of 0.04, and FDR correction for multiple testing (--step 2000, --minBoundaryDistance 5000 --pvalue 0.01 --delta 0.04 --correctForMultipleTesting fdr). We selected these parameters to ensure that we recover a similar number of TADs as previously reported (Chathoth and Zabet, 2019). Finally, we called strong TAD borders using a stringent value of the threshold of the difference between the TAD separation score of 0.08. This value ensured that we retrieved the strongest half of TADs. The enriched contacts were extracted with HiCExplorer using the observed/expected ratio method.
Chromatin loops: Chromatin loops were called with the HICCUPS tool from the Juicer software suite (Durand et al. 2017) on all mutants as done previously (Chathoth and Zabet, 2019). Loops were called using a 2 kb resolution, 0.05 FDR, Knight-Ruiz normalisation, a window of 10, peak width of 5, thresholds for merging loops of 0.02,1.5,1.75,2 and distance to merge peaks of 20 kb (-k KR -r 2000 -f 0.05 -p 5 -i 10 -t 0.02,1.5,1.75,2 -d 20000).
RNA-seq analysis: Reads were first trimmed using Trimmomatic (v0.39) (Bolger et al., 2014) and then aligned to Drosophila melanogaster (dm6) genome (Adams et al., 2000; dos Santos et al., 2015) using TopHat(v2.1.2) (Kim et al., 2014) with Bowtie2(v2.3.4.1) (Langmead et al., 2012) (Table S3). Finally, we used Picard tools (http://broadinstitute.github.io/picard/) to deduplicate reads and HTseq (Anders et al., 2015) to count reads.
Genome_build: dm6
Supplementary_files_format_and_content: BED files contain the coordinates of TADs; bepe files contain the coordinates of chromatin loops
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| Submission date |
Mar 16, 2020 |
| Last update date |
Feb 07, 2022 |
| Contact name |
Nicolae Radu Zabet |
| E-mail(s) |
nzabet@essex.ac.uk
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| Phone |
+44(0)1206872630
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| Organization name |
University of Essex
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| Department |
School of Life Sciences
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| Street address |
School of Life Sciences, University of Essex,
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| City |
Colchester |
| ZIP/Postal code |
CO4 3SQ |
| Country |
United Kingdom |
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| Platform ID |
GPL25244 |
| Series (1) |
| GSE147059 |
The role of insulators and transcription in 3D chromatin organisation of flies |
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| Relations |
| BioSample |
SAMN14384299 |
| SRA |
SRX7918788 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4413972_htseq_RNAseq_BG3_BEAF32_KD_rep2.txt.gz |
67.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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