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Sample GSM5852374

Query DataSets for GSM5852374

Status

Public on Dec 31, 2022

Title

ChIP_MEF_Ola_H3K4me3_IFNb_0h_Rep1

Sample type

SRA

Source name

Mouse embryonic fibroblasts

Organism

Mus musculus

Characteristics

cell type: Mouse embryonic fibroblasts
strain: 129/Ola
treatment: IFNb_0h
chip antibody: ab8580; abcam; #lot GR188707-1

Treatment protocol

All cells were treated with 500U/ml of self-made interferon beta (16.6U/µl) for 1 hour or 6 hours

Growth protocol

Embryonic stem cells: Cultured on 0.1 % gelatinized tissue flasks in ESC media. Cells were incubated at 37 °C and 5 % CO2. Media was PowerStem ESPro1 (PAN-Biotech; P04-77510K) with self-made LIF (1:1000).
Mouse embryonic fibroblasts: Cells were incubated at 37 °C and 5 % CO2. Media was DMEM (Gibco, 11880-28), 10% FCS (PAN-Biotech; P30-3602), 1x L-Glutamine (PAN-Biotech, P04-80050), 1x PenStrep (PAN-Biotech, P06-07050).
Neural progenitor cells: ESCs were differentiated into NPCs based on the Bible-protocol Bibel et al. 2007. 4-5*10^6 cultured ESCs were split onto a T75 UltraLow-BindingPlates (Corning, 3814) in 15 ml StemPAN media (PAN-biotech, P08-50500). After 4 days 5µM retinoic acid (Sigma-Aldrich, R2625) was added. On day 8, cells were platted on plates coated with 1% Matrigel (Corning, 356230) in neuronal base medium (Gibco, 211103049), G5 supplements (Gibco, 17503012), NSC supplements (Gibco, 17502048). Experiments were performed five days after neuronal plating.

Extracted molecule

genomic DNA

Extraction protocol

ChIP-seq for histone marks in MEFs: Cells were crosslinked by 1 % Formaldehyde for 10 min at room temperature and stopped with 125 mM Glycine for 5 min. Samples of around 40 million cells were treated with 40 U of MNase for 15 min at 37 °C. The samples were shared for 15 min (Burst 200; Cycle 20%; Intensity 8). Replicate 1 was shared for 30 min and no MNase treatment was performed. 4µg of antibody was used per sample and incubated for 2 hours at 4 °C. Protein G-beads were added over night. Multiple low and high salt washes were performed and the samples were eluted. Samples were reverse crosslinked and stored at -20°C.
ChIP-seq for histone marks in MEFs: Libraries were made using NEBNext Ultra™ II DNA Library Prep Kit for Illumina (New England Biolabs, E7645)

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

ChIP bed files: Raw reads were mapped with Bowtie (v1.2.2). The mapped reads were sorted, duplicates and reads in blacklisted regions removed with bedtools (v2.27.1).
ChIP Norm counttables: Bam files were used to count reads around peaks of STAT1p701 and STAT2 by bedtools (v2.27.1). Reads were normalized by sequencing depth, fragment length and enrichment to control. For histon marks H3 signal was the control. For STAT ChIPs IgG_Rb_CST.
Genome_build: mm10

Submission date

Feb 01, 2022

Last update date

Dec 31, 2022

Contact name

Markus Muckenhuber

E-mail(s)

m.muckenhuber@dkfz.de

Organization name

DKFZ

Lab

AG Rippe