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Sample GSM2754205

Query DataSets for GSM2754205

Status

Public on Nov 06, 2017

Title

UV_IgG_control

Sample type

SRA

Source name

germinal center B-cell

Organism

Homo sapiens

Characteristics

cell type: B cell lymphoma
cell line: OCI-LY1
chip antibody: sc-2027, Lot# D1513

Treatment protocol

UV laser irradiation of living cells was performed using Quanta-Ray pulsed Nd:YAG laser (Model GCR-150, Spectra Physics) equipped with an HG-2 harmonic generator (Spectra Physics) and dichroic mirrors (DHS-2 Quanta-Ray dichroic harmonic separator) to give monochromatic light at 266 nm. The laser beam was focused by a fused silica lens, deviated by 90° and adjusted to fit the surface of the sample area. The laser energy at the sample position was determined using a Power/Energy Meter (Nova, Ophir Optronics Ltd.) equipped with a Power Thermal Sensor (Model 10A-P-SH, Ophir Optronics Ltd.). The parameters for UV laser irradiation were as follows: pulse duration 5 ns, repetition rate 10 Hz, energy per pulse 50 mJ, diameter of the laser beam 6 mm. Formaldehyde (FA) ChIP was performed as described. OCI-Ly1 cells were fixed with 1% FA for 10 min under rotation at room temperature and the reaction was quenched by addition of 125 mM glycine for 5 min.

Growth protocol

The germinal center B cell like diffuse large B-cell lymphoma (DLBCL) OCI-Ly1 cell line (GCB-DLBCL OCI-Ly1) was maintained in Iscove’s Modified Dulbecco’s Medium (Gibco) supplemented with fetal calf serum (10% v/v, Gibco) at 37°C and 5% CO2.

Extracted molecule

genomic DNA

Extraction protocol

Immediately after irradiation, OCI-Ly1 cell suspensions were added to ice cold PBS (pH 7.4) containing protease inhibitors according to the manufacturer’s recommendations (Complete Protease Inhibitor, Roche Applied Science). All subsequent steps were performed at 4°C. After centrifugation at 1,000 g for 7 min, the pellet was resuspended in 250 µl of ChIP lysis buffer (0.5% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, protease inhibitors). Chromatin was sheared on ice (Microtip sonicator, Labsonic U, Sartorius) into fragments averaging 250 bp and centrifuged at 16,000 g for 10 min. Fragmentation of chromatin was analyzed with purified DNA using agarose gel electrophoresis and a 2100 Bioanalyzer (Agilent Technologies). An aliquot of sheared chromatin was used for UV input DNA sequencing. The supernatant was pre-cleared for 1 h using pre-washed protein A magnetic beads according to the manufacturer’s instructions (Dynabeads Protein A, Invitrogen). A ChIP-grade anti-Bcl6 antibody (Bcl-6 (N-3), sc-858, Lot# A3013, Santa Cruz) was used for ChIP. The specificity and suitability for ChIP was confirmed by immunoblot analysis. For UV IgG control ChIP, normal rabbit IgG (sc-2027, Lot# D1513, Santa Cruz) was used. Clarified chromatin extracts were incubated for 12 h with specific anti-Bcl6 antibody and control IgG antibody, respectively. Nucleoprotein-antibody complexes were precipitated using pre-washed beads for 1 h. The supernatant was removed and beads were sequentially washed twice in low salt (0.1% SDS, 1% Triton-X 100, 150 mM NaCl, 20 mM Tris pH 8.0, 2 mM EDTA), high salt (0.1% SDS, 1% Triton-X 100, 500 mM NaCl, 20 mM Tris-HCl pH 8.0, 2 mM EDTA), LiCl (0.5% NP-40, 0.5% deoxycholic acid (DOC), 250 mM LiCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) and TE (10 mM Tris pH 8.0, 1 mM EDTA) buffer. For elution of immunoprecipitated nucleoprotein-antibody complexes, beads were resuspended in 0.1 M citrate buffer (pH 2.2) for 2 min at room temperature. The supernatant was removed and transferred into Tris-HCl (pH 8.0) for neutralization. After RNase A treatment (50 µg/ml, Fermentas) for 1 h at 37°C and Proteinase K treatment (100 µg/ml, Fermentas) for 12 h at 50°C, genomic DNA was purified by phenol/chloroform/isoamylalcohol extraction and ethanol precipitation. Formaldehyde (FA) ChIP was performed as described except for the following steps. Cells were harvested, washed twice with ice cold PBS (pH 7.4) and lysed in RIPA buffer (1% NP-40, 0.5% DOC, 0.1% SDS, 150 mM NaCl, 50 mM Tris pH 8.0, 5 mM EDTA, protease inhibitors). For elution of immunocomplexes, beads were resuspended in elution buffer (1% SDS, 0.1 M NaHCO3) and crosslinking was reverted by addition of 0.3 M NaCl and incubation for 5 h at 65°C.
Sequencing libraries were prepared from UV-ChIP, FA ChIP and control DNA fragments according to the manufacturer’s protocol (NEBNext® Ultra™ DNA Library Prep Kit). In brief, DNA fragments were end-repaired and the blunt, phosphorylated ends were treated with Klenow DNA polymerase and dATP to yield a 3’ A base overhang for ligation of adapters. After adapter ligation, DNA was PCR amplified (15 cycles). Libraries were size-selected to achieve ChIP DNA fragment lengths of 200-300 bp. The purified DNA was captured on an Illumina flow cell for cluster generation and libraries were sequenced on a HiSeq 2500 (Illumina) instrument according to manufacturer's protocol.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Data processing

Reads were mapped onto the human genome reference (GRCh37/hg19) using Bowtie (-l 50 -3 1 -n 2 -m 1 --best --strata)
Uniquely mapped reads including a maximum of two mismatches were accepted and redundant reads with identical coordinates were filtered out.
Reads were screened against the blacklist regions (collection of signal artifacts) in the human genome (https://sites.google.com/site/anshulkundaje/ projects/blacklists) and overlapping reads were removed.
Genome_build: GRCh37 (hg19)
Supplementary_files_format_and_content: Read density profiles (signal tracks) were generated as pileup signal files (normalized by fragment pileup in million reads) (q-value 5e-2) and fold enrichment (FE) tracks were generated by normalized ChIP over input DNA pileup signal files using MACS2.

Submission date

Aug 25, 2017

Last update date

May 15, 2019

Contact name

Arndt Steube

Organization name

Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute (HKI)

Department

Cell and Molecular Biology

Lab

HPSaluz Lab