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Sample GSM4671251

Query DataSets for GSM4671251

Status

Public on Apr 14, 2021

Title

C_R2

Sample type

SRA

Source name

U2OS cells

Organism

Homo sapiens

Characteristics

cell line: U2OS
overexpressed protein: None
spike-in: YES
antibodies: FLAG: Sigma, F7425; H2Av: Active Motif, 39715

Treatment protocol

Protein expression was induced in media with Tetracycline (2 µg/ml, 48 hrs) in DMEM, 10 % FBS and 1 % P/S.

Growth protocol

U2OS were grown in DMEM with 10 % heat-inactivated FBS and 1 % penicillin/streptomycin under Blasticidin (5 µg/ml) selection and additionally Hygromycin B (100 µg/ml) selection for cell lines with pcDNA5-FRT-TO integration at 37 °C with 5 % CO2. Drosophila S2 cells (male) were grown in M3+BPYE media: Shields and Sang M3 Insect Medium, KHCO3, yeast extract, bactopeptone, 10 % heat-inactivated FBS and 1X penicillin/streptomycin at 25 °C with 5 % CO2.

Extracted molecule

genomic DNA

Extraction protocol

Cells were processed using the truChIP Chromatin Shearing Kit (Covaris, 520127) following manufacturer’s instructions. 20 million nuclei were sonicated in 1mL tubes using a Covaris M220 to obtain chromatin frgament size of 250-500bp. Sonicated chromatin was centrifuged at 14,000 rpm at 4ºC for 10 minutes and the supernatant was isolated for subsequent steps. In parallel, Drosophila S2 cells were ﬁxed, lysed and sonicated as described above. U2OS input chromatin was mixed with Drosophila S2 chromatin (1.5% of total chromatin) after sonication. Chromatin associated to DNAJC9-MYC-Flag was obtained by incubating sonicated chromatin with anti FLAG antibody.
Libraries were costructed using the KAPA Hyperprep kit following manufacturer’s instruction, except that 1.25 µM of Illumina compatible indexed adapters (Pentabase) were ligated to A-tailed DNA.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Data processing

Illumina RTAv2 software was used for basecalling
Raw reads were trimmed using Trim Galore! (Galaxy version 0.4.2) to remove low-quality reads and adaptor sequence.
Libraries were mapped to the hg38 and dm3 genome assemblies using Bowtie2 (Galaxy version 2.2.6.2).
Duplicates and reads mapping to the Broad Institute's blacklist (Encode Project Consortium, 2012) were discarded. Remaining reads were extended by 250 bp for downstream analyses. Replicates were merged for plotting and downstream analyses.
Genome_build: hg38 and dm3
Supplementary_files_format_and_content: Bedgraph files contain either RPM, RRPM or RRPM, n+1, log-transformed read counts for merged replicates (R1+R2) of ChIP-seq datasets from 10 Kb windows with a 5 Kb step.

Submission date

Jul 14, 2020

Last update date

Apr 14, 2021

Contact name

Anja Groth

E-mail(s)

anja.groth@cpr.ku.dk

Organization name

Novo Nordisk Foundation Center for Protein Research

Street address

Blegdamsvej 3B