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Sample GSM2584449

Query DataSets for GSM2584449

Status

Public on Jun 26, 2017

Title

Beaf-32 + M1BP RNAi Hi-C biological rep B technical rep 1

Sample type

SRA

Source name

S2_M1BP_Beaf-32_RNAi

Organism

Drosophila melanogaster

Characteristics

cell line: S2
bio_replicate: 2
technical_rep: 1

Treatment protocol

For knockdown experiments, 7.5 million cells transfected with 100μg of dsRNA on 10cm dishes, using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific). The M1BP knockdown lasted for 7 days while that of Beaf-32 lasted for 4 days. For M1BP-Beaf-32 double knockdown, cells were first treated with M1BP dsRNA at day 1, followed by treatment with Beaf-32 dsRNA at day 3, and were collected at day 7. All knockdown experiments were carried out in duplicate.

Growth protocol

Drosophila S2 cells were cultured in Express Five SFM (Thermo Fisher Scientific) supplemented with glutamax, at 27°C at a density of 1-16 million/ml.

Extracted molecule

genomic DNA

Extraction protocol

S2 cells have been fixed with 2% formaldehyde for 10 minutes at room temperature. Glycine at 125 mM final concentration has been added to the plates followed by 5 minutes incubation. After two washes in PBS, cells have been scraped off the plate and pelleted. Each pellet (10-50 million of cells) has been resuspended in 1 ml of lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% IGEPAL CA-630) and nuclei have been extracted by sonication (Arrigoni et al. 2016) using Covaris E220 sonicator (settings: 75W peak power, 2% duty factor, 200 cycles/burst), for 60-90 seconds until about 70% of intact nuclei have been released.
10-50 ng of DNA bound to beads have been used for library preparation using a modification of the NEBNext Ultra II DNA library preparation workflow (NEB, E7645). DNA bound to beads have been end-repaired, A-tailed, adaptor-ligated using manufacturer’s instruction. Beads were reclaimed on a magnet, washed once in EB and eluted at 98 °C for 10 minutes. DNA has been USER-treated to open the adapters in meantime to PCR amplification.

Library strategy

Hi-C

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 3000

Description

[processed data file:]
Beaf32_M1BP_KD_merge_rf_corrected_score.bedgraph

Data processing

mapping : each mate aligned separately to drosophla genome (dm3, ensembl) using bwa mem with parameters ` -E50 -L0`
build hic matrix : using hicBuildMatrix (HiCExplorer v1.7.0, using restriction fragment resoltiion) with parameters `-rs <dpn2.bed> `
correct hic Matrix : using hicCorrectMatrix (HiCExplorer v1.7.0)
find TADs : using hicFindTADs (HiCExplorer v1.7.0) with parameters `TAD_score --minDepth 10000 --maxDepth 40000 --step 2500` followed by `find_TADs --pvalue 0.001 --delta 0.01`
Genome_build: dm3
Supplementary_files_format_and_content: bedgraph format. The bedgraph files contain TAD separation scores in windows over the genome, calculated using HiCExplorer. A HiC matrix was created for each sample using HiCExplorer and matrices for replicates for each sample were merged. Matrices were then normalized using ICE correction and TAD separation score was calculated in windows.

Submission date

Apr 19, 2017

Last update date

May 15, 2019

Contact name

Thomas Manke

E-mail(s)

[email protected]

Organization name

Max Planck Institute of Immunobiology and Epigenetics

Street address

Stuebeweg 51