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Sample GSM3500766

Query DataSets for GSM3500766

Status

Public on Apr 25, 2019

Title

D1-10_input_Rep1

Sample type

SRA

Source name

mESCs-Rb TKO

Organism

Mus musculus

Characteristics

cell type: ESC
genotype: Rb TKO

Growth protocol

mESC lines were maintained feeder-free on 0.2% gelatin-coated (Sigma G9382) plates in Dulbecco’s modified Eagle’s medium (Gibco SH30243.01) containing 15% fetal bovine serum (Hyclone SH30071.03; VWR Seradigm 97068-085), MEM non-essential amino acids (Gibco 11140-050), penicillin-streptomycin-glutamine (Gibco 10378-016), and 0.1 mM beta-mercaptoethanol (Gibco 21985-023), and supplemented with LIF. Cells were passaged enzymatically using 0.05% Trypsin-EDTA (Gibco 15400-054). Single-cell suspensions of 1000 mESCs/mL were cultured in low density assays.

Extracted molecule

genomic DNA

Extraction protocol

Chromatin immunoprecipitation was done according to a published method (O’Geen, H., Nicolet, C.M., Blahnik, K., Green, R. & Farnham, P.J. Comparison of sample preparation methods for ChIP-chip assays. Biotechniques 41, 557-580 (2006). Briefly, mESCs were crosslinked with 1% formaldehyde in PBS for 10 min at room temperature and the reaction was stopped by adding glycine to final concentration of 1.25M for 5 minutes at room temperature. Crosslinked cells were collected with a cell scraper, rinsed twice with PBS, and resuspended at a concentration of 5 x 10^7 cells/mL of swelling buffer (0.1 M Tris pH 7.6, 10 mM KOAc, 15 mM MgOAc, 1% NP40) to remove the cytoplasmic fraction. Nuclei were resuspended at a concentration of 10 x 10^8 cells/mL of nuclei lysis buffer (50 mM Tris-Cl pH 8.0, 10 mM EDTA, 1% SDS), and lysate was sonicated for 90 cycles (30 sec on / 50 sec off) in a Diagenode Bioruptor (Diagenode UCD-300). The sonicated chromatin was precleared by adding washed and blocked StaphA at a concentration of 10 uL/1 x 10^7 cells (Calbiochem 507862). StaphA were prepared as previously described, washed in dialysis buffer (2 mM EDTA, 50 mM Tris-Cl pH 8.0), and blocked in 10 mg/mL salmon sperm DNA (EMD Millipore 16-157) and 10 mg/mL BSA. For each IP, 100 uL of precleared chromatin was diluted in twice the volume of IP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 167 mM NaCl) and 5 ug of antibody was added. Antibodies against H3K4me3 (Abcam ab8580), H3K9Ac (Active Motif 39137) were used.
Libraries were constructed using the NEBNext DNA Sample Prep Master Mix Set for Illumina (NEBNext E6040) and NEBNext Multiplex Oligos for Illumina (NEBNext E7335 and E7500), according to kit instructions. Libraries were then quantified using the KAPA Library Quantification Kit with Universal qPCR Master Mix (KAPA Biosystems, KK4824) according to kit instructions, and quantitative PCR was performed using the SYBR GreenER MasterMix (Invitrogen) on an ABI 7900HT Detection System. Pooled samples were sequenced on a HiSeq4000.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 4000

Data processing

Obtained reads were mapped to mouse reference genome mm9 with bowtie2.3.4. Peakcalling was performed with MACS2.1.1 using broadpeaks, merging the two replicas from identical cell lines, and using the merged input samples as control. Differential peaks were determined with the R package DiffBind, again merging the two replicas from the identical cell line and using the peaks from MACS.
Genome_build: mm9
Supplementary_files_format_and_content: excel file with differential bound regions from diffbind

Submission date

Dec 04, 2018

Last update date

Apr 25, 2019

Contact name

Julia Arand

E-mail(s)

juliaarand@googlemail.com

Organization name

Medical University of Vienna

Street address

Schwarzspanierstr. 16