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Sample GSM2230078

Query DataSets for GSM2230078

Status

Public on Aug 02, 2016

Title

1.K4R-H3-K36me3

Sample type

SRA

Source name

yeast cells

Organism

Saccharomyces cerevisiae

Characteristics

strain: H3K4R
library: Histone mutation library
1st ip: H3
2nd ip / input: K36me3

Growth protocol

Yeast cells were grown in YPD media at 30ºC with constant shaking to OD 0.6-0.8.

Extracted molecule

genomic DNA

Extraction protocol

Cells were fixed with 1% formaldehyde for 15 minutes at room temperature with occasional shaking, quenched with 0.125 M glycine for 5 minutes at room temperature with occasional shaking, collected by centrifugation, (4000 g, 5 minutes), washed with cold ddH2O supplemented with EDTA-free protease inhibitors cocktail (Roche) and the pellet was resuspended in buffer Z (1 M sorbitol, 50 mM Tris 7.4, 10 mM β-mercaptoethanol) with zymolyase (Seikagaku) at 0.3 - 1 units per 1 ml of original cell volume. Cells were gently rotated at 30ºC for 25 minutes until > 95% of cells were spheroplasted. Spheroplasts were pelleted (6500 g, 10 minutes) and resuspended in NP buffer (10 mM Tris pH 7.4, 1 M sorbitol, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, and 0.075% NP-40, freshly supplemented with 1 mM β-mercaptoethanol, 500 μM spermidine, and EDTA-free protease inhibitor cocktail) at final concentration of 200 OD/ml. Chromatin was digested with 12.5 units/ml MNase (Worthington) for 20 minutes at 37ºC, and digestion was stopped by removing the tubes into ice and addition of 1 volume of ice cold MNase stop buffer ( 220 mM NaCl, 0.2% SDS, 0.2% DOX, 10 mM EDTA, 2%,Triton X-100, EDTA-free protease inhibitor cocktail). Tubes were kept on ice for 10 minutes, vortexed 3 x 10 seconds, centrifuged (16,000 g, 10 minutes, 4ºC), and the supernatant containing the nucleosomes was removed to a fresh tube.
See attached paper - http://biorxiv.org/content/early/2016/06/27/060962

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Data processing

Pair-end reads were mapped to the yeast genome (sacCer3) using bowtie2 with maximal fragment size of 1000bp.
We treated duplicate fragments as potential PCR artifacts. We thus treated the set of unique fragments found as the read-set. We defined mononucleosome fragments as these shorter than 220bp.
We used the nucleosome location atlas defined by Weiner et al(Weiner et al., 2015). We measured nucleosome coverage by counting the number of fragments overlapping a window of size 50bp around the center of the nucleosome.
Genome_build: sacCer3
Supplementary_files_format_and_content: csv file containing read counts for each nucleosome location in each combinatorial ChIP sample

Submission date

Jul 11, 2016

Last update date

May 15, 2019

Contact name

Nir Friedman

E-mail(s)

nir@cs.huji.ac.il

Organization name

The Hebrew University of Jerusalem

Department

School of Engineering