GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5321297

Query DataSets for GSM5321297

Status

Public on Oct 12, 2021

Title

Input_wildtype_2

Sample type

SRA

Source name

whole cell

Organism

Schizosaccharomyces pombe

Characteristics

genotype: WT
chip antibody: none (Input)

Extracted molecule

genomic DNA

Extraction protocol

100 mL yeast cultures were grown to mid-log phase to OD600 = 0.6. The cultures were cooled down at RT for 10 min and fixed with 1% FA for 20 min at RT on the shaker. Cross-linking was stopped with 150 mM Glycine for 10 min at RT. Cells were washed 2x with 30 mL ice-cold dH2O, the pellet was frozen in liquid nitrogen and kept at -80°C until further processing. Pellets were resuspended in 1 mL FA(1) buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 (v/v), 0,1% NaDeoxycholate (w/v), 0,1% SDS (w/v), supplemented with Roche protease inhibitors. Cells were broken in a bead beater (Precellys): 9x30s. After centrifugation, the supernatant and the pellet were sonicated for 15 min at 4°C. Chromatin extracts were spun down for 10 min at 14000 rpm at 4°C. 500 µl chromatin was used and 1 µl of H3K9me2 antibodies (Abcam, Ab1220). Samples were incubated with antibodies O/N at 4°C. 25 µl of FA(1)-washed Dynabeads were added to each sample and they were incubated for 2 hours at 4°C. Samples were then washed 3x for 5 min at RT with FA(1) buffer, FA(2) buffer (FA(1) buffer with 500 mM NaCl), once with LiCl buffer (10 mM TrisHCl, pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0,5% NP-40 (v/v), 0,5% NaDeoxycholate (w/v)) and once with TE buffer. DNA was eluted from the antibodies with ChIP Elution buffer (50 mM Tris HCl, pH 7.5, 10 mM EDTA, 1% SDS) for 15 min at 65°C. DNA was treated with RNAseA, Proteinase K and de-crosslinked O/N at 65°C. DNA was purified with Zymo Research ChIP DNA Clean and Concentrator kit according to the manual instructions. To obtain enough material for the library preparation three technical IP replicates were pulled.
ChIP-seq libraries were prepared with 2 ng of DNA with NEBNext Ultra II DNA Library Prep Kit for Illumina according to the manual instructions. The libraries were sequenced at LAFUGA at the Gene Center (LMU).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

Single-end reads (50 bp) were mapped to the reference genome (Schizosaccharomyces pombe ASM294v2) using bowtie2 (version 2.2.9).
Reads were counted in either 250 bp or 10 kb windows (bins) using the windowCounts() function from the csaw R/Bioconductor package (version 1.18.0). Normalization factors were calculated by the normFactors() function using the “TMM” method on count matrices with 10 kb bins and applied on count matrices with 250 bp bins. Normalized, log2-transformed count matrices were obtained by the cpm() function (edgeR package, version 3.26.8). ChIP samples were normalized by their corresponding inputs (i.e. subtraction in log2-scale). Input-normalized count matrices were converted to coverage vectors using the coverage() function (GenomicRanges package, version 1.38.0) and exported as bigwig files (rtracklayer package, version 1.44.4).
genome build: Schizosaccharomyces pombe ASM294v2
processed data files format and content: Bigwig format. Normalized coverage.

Submission date

May 18, 2021

Last update date

Oct 12, 2021

Contact name

Tamas Schauer

E-mail(s)

[email protected]

Organization name

Helmholtz Zentrum München

Department

Institute of Epigenetics and Stem Cells