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Sample GSM3475690

Query DataSets for GSM3475690

Status

Public on Jan 18, 2019

Title

BG3 rep1

Sample type

SRA

Source name

BG3 cells

Organism

Drosophila melanogaster

Characteristics

cell line: BG3 cells

Growth protocol

Kc167 and BG3 cells were grown to 80-90% confluence in Schneider’s Drosophila medium with 5%FBS or 10% FBS respectively and antibiotics (Pen-Strep) in 100 mm cell culture plates.

Extracted molecule

genomic DNA

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).

Library strategy

Hi-C

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Data processing

HiSeq Control Software HD 3.4.0.38 was used for basecalling (for Kc167 smaples) and RTAVersion 2.7.7 was used for basecalling (for BG3 smaples) .
Each pair of the PE reads were aligned separately to Drosophila melanogaster (dm6) genome using BWA-mem (version 0.7.15-r1140) with options -t 20 -A1 -B4 -E50 -L0. HiCExplorer (version 2.1.1) was used to build and correct the contact matrices and detect TADs. The contact matrices were built at the DpnII restriction sites. Using a minimum allowed distance between restriction sites of 150 bp and a maximum distance of 1 Kb. We merged the two BG3 biological replicates and the two Kc167 replicates. The matrices were corrected using the thresholds (-1.4 and 5). 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). Finally, we called strong TAD borders using a stringent value of the threshold of the difference between the TAD-separation score of 0.08. Since, the Kc167 cell line is a female derived cell line and BG3 is a male derived cell line, we excluded sex chromosomes from our analysis.
Chromatin loops were called with the HICCUPS tool from the Juicer software suite (version 1.7.6). 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).
Genome_build: dm6
Supplementary_files_format_and_content: BED files contain the coordinates of TADs; bepe files contain the coordinates of chromatin loops

Submission date

Nov 15, 2018

Last update date

Jan 18, 2019

Contact name

Nicolae Radu Zabet

E-mail(s)

nzabet@essex.ac.uk

Phone

+44(0)1206872630

Organization name

University of Essex