DNaseI digestion of T. kodakarensis chromatin - SRA

SRX315137: DNaseI digestion of T. kodakarensis chromatin
1 ILLUMINA (Illumina HiSeq 2000) run: 161.4M spots, 31G bases, 20Gb downloads

Design: T. kodakarensis strain KOD1 was cultivated under anaerobic conditions at 85°C in nutrient-rich medium ASW-YT, supplemented with 0.2% elemental sulfur (0.8x ASW-YT-S0) as described (Maruyama et al., 2011). Cells were harvested from late log-stationary phase culture and cell suspensions (1010 cells per milliliter) were disrupted by the addition of extraction buffer (25 mM HEPES [pH 7.0], 15 mM MgCl2, 100 mM NaCl, 0.4 M Sorbitol, and 0.5% Triton X-100). After incubating for 10 min at 4°C, soluble proteins (supernatant) and a chromosomal DNA-enriched insoluble fraction (pellet) were separated by centrifugation at 14,000 g for 20 min. The insoluble chromatin fraction, containing DNA and chromatin-associated protein, was washed with the extraction buffer and frozen at -20°C until use. Chromatin pellet was digested with 0.1 units of DNaseI (Worthington Biochemical Corp., NJ, USA) for 1 hour at 37°C in the presence of 10 µg/µl RNase A as described (Maruyama et al., 2011). Resulting DNA was treated with Proteinase K (1 mg/ml) for 2 hours at 37°C and purified by phenol-chloroform extraction and ethanol precipitation. DNA fragments ranged from 10-100bp. Adapters (Illumina) were ligated to 4 µg of DNA fragments using the NEBNext DNA sample prep master mix set 1 and size selected on polyacrylamide gels to preserve the size distribution of the fragments (Kent et al., 2011). Ligated fragments were amplified (12 cycles) using Phusion DNA polymerase and adaptor-specific primers (Illumina), followed by purification using AMPure XP beads (Agencourt). 7 pM DNA was hybridized on one lane of an Illumina flowcell resulting in a clusters density of ~700 K/mm2 PF and sequenced using 100 nucleotide paired-end mode on an Illumina HiSeq 2000 using TruSeq SBS reagents version 3.

Submitted by: SCHOOL OF BIOSCIENCES, CARDIFF UNIVERSITY

Study: Thermococcus kodakarensis KOD1 Epigenomics

PRJNA209412 • SRP045453 • All experiments • All runs

show Abstracthide Abstract

Chromatin sequencing technology was used to analyse the genomic structure of this euryarchaeal model. We discovered positioned chromatin particles throughout the genome that appear to consist of variably sized aggregates of the archaeal histone dimer. We therefore describe a novel form of beads-on-a-string chromatin.

Sample: Generic sample from Thermococcus kodakarensis KOD1

SAMN02212697 • SRS452611 • All experiments • All runs

Organism: Thermococcus kodakarensis KOD1

Library:

Instrument: Illumina HiSeq 2000

Strategy: DNase-Hypersensitivity

Source: GENOMIC

Selection: DNase

Layout: PAIRED

Spot descriptor:

1 forward101 reverse

Runs: 1 run, 161.4M spots, 31G bases, 20Gb

Run	# of Spots	# of Bases	Size	Published
SRR922437	161,376,537	31G	20Gb	2013-09-15

ID:: 440271

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