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Sample GSM1556337

Query DataSets for GSM1556337

Status

Public on Feb 18, 2016

Title

p52_ChIPSeq_rep1

Sample type

SRA

Source name

Hodgkin Lymphoma Cell Line

Organism

Homo sapiens

Characteristics

cell line: L1236
dsmz no: ACC 530
chip antibody: NFkB p52 Millipore #05-361, mouse mk Ab

Growth protocol

90% RPMI + 10% heat inactivated FBS

Extracted molecule

genomic DNA

Extraction protocol

Cells were fixed with 1% Formaldehyde, lysed with 50 mM Tris-HCl, pH 8, 5mM EDTA, 1% SDS, sonicated with the Bioruptor (12 cycles), Setting M, 30 sec sonication/30 sec break per cycle. Immunoprecipitation and DNA-purification was performed according to the Upstate protocol.
Libraries were prepared using Illumina's ChIP-Seq Sample Prep Kit (#IP-102-1001) according to the instructions of the manufacturer. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, size selection of the library was performed by excision of the region from 175 to 225 bp. DNA was PCR amplified with Illumina primers for 18 cycles. The purified DNA was captured on an Illumina flow cell for cluster generation. Each library was validated using an Agilent 2100 bioanalyzer and sequenced on the Genome Analyzer following the manufacturer's protocols.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer II

Data processing

Base-calling with Illumina Casava pipeline
Alignment with bowtie retaining only uniquely mapping reads
Technical replicates were merged after alignment and likely PCR artifacts were removed using samtools
To obtain comparable total read counts we downsampled the merged bam files to a total read count of 10Mio reads
Peak calling on each biological replicate was performed with macs. Then biological replicates were used to estimate the irreproducible discovery rate (IDR). After IDR analysis reads from biological replicates were merged and jointly analysed with MACS. Final peak calls were defined as regions with IDR < 0.05 and MACS FDR < 0.05.
Overlaps between peaks of different libraries were investigated as follows. First we defined the union of all regions bound in any of the experiments and then determined overlaps with peak calls from each of the individual libraries. Peaks were annotated to the closest gene and the closest TSS within a window of 1Mb according to the annotation of Ensembl version 54.
Genome_build: hg18
Supplementary_files_format_and_content: Tab separated text containing the coordinates of the peak regions. In addition for each NFKB subunit there are the following columns: indicator variable for a peak call, IDR, Macs FDR, fold enrichment over input, read pileup and peak summit.

Submission date

Dec 01, 2014

Last update date

May 15, 2019

Contact name

Marina Kolesnichenko

E-mail(s)

marina.k@oxfordalumni.org

Organization name

Max-Delbrück-Center

Lab

Signal Transduction in Tumor Cells