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| Status |
Public on Jun 01, 2015 |
| Title |
Sample_Input_Index_5_lane3 |
| Sample type |
SRA |
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| Source name |
Diffuse large B-cell lymphoma (DLBCL) cell line OCI-Ly4
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| Organism |
Homo sapiens |
| Characteristics |
cell line: OCI-Ly1
chip antibody: input
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| Treatment protocol |
Immunoprecipitation using anti-histone H3 trimethyl K4 (ab8580, Abcam, Cambridge, MA) was performed in crosslinked sonicated extracts. Cells were grown at 1-1.5 million per ml and protein and DNA were crosslinked for a 10 minute treatment with 1% Formaldehyde (Thermo Scientific, Rockford, IL) and harvested. 100 x 106 cells were lysed by flash freezing in liquid N2 and stored at -80ºC. Chromatin shearing was performed using a Covaris S220 sonicator (Covaris, Woburn, MA) using the following conditions: 1 ml tubes with approximately 30 million cells in buffer containing 0.1% SDS (Covaris Buffer D3) were sonicated using peak intensity power of 140, duty factor of 5.0 and 200 cycles per burst, for 18 minutes. Extent of shearing was monitored with a 1% agarose gel and confirmed by separation on an 2100 High sensitivity Bioanalyzer chip (Agilent Technologies, Santa Clara, CA) at the completion of the immunoprecipitation. Immunoprecipitation was carried out at 4ºC with overnight incubation using 97.5 x 106 cell equivalents of chromatin and 15ug of antibody. Immunoprecipitates were captured with Protein A Dynabeads (Novex Life Technologies,Grand Island, NY), washed, and resuspended in buffer containing 100ug of RNAse A and 20ug of PCR grade Proteinase K (Qiagen, Germantown, Maryland). Crosslinks were reversed by incubation at 65ºC in the presence of 1% SDS and 0.3M NaCl.
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| Growth protocol |
Diffuse large B-cell lymphoma (DLBCL) cell line OCI-Ly1 was grown in medium containing 90% Iscove's, 10% fetal calf serum and supplemented with penicillin G and streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was then subjected to phenol:chloroform extraction followed by purification using a PCR purification kit (Qiagen, Germantown, Maryland). Quantitative real time PCR (qRT-PCR) on positive housekeeping genes was performed on both input and eluted chromatin, to validate the ChIP efficacy.
Libraries compatible with Illumina TruSeq adapter sequencing (Illumina Inc, San Diego, CA) were made as follows: 10 ng of either immunoprecipitated or input DNA were end-repaired using 3 units of T4 DNA polymerase, 1 unit of Klenow DNA polymerase and 10 units of T4 DNA polymerase with 30 minutes incubation at 20ºC; A-tailing was performed with 5 units of Klenow fragment and 10mM dATP (New England Biolabs, Ipswich, MA) for 30 minutes at 37ºC. 2 ul of 1uM of annealed Illumina TruSeq adapters (Integrated DNA Technologies, Coralville, IA) were used in an overnight ligation at 4ºC with 2000 units of T4 DNA Ligase. To account for the migration pattern of Y-forked Illumina adaptors ligated products from 250-350bp were size selected in a 1% agarose gel. After purification, the PCR reaction was carried out with 300uM dNTP, 200uM of primers and 1 unit of Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Rockford, IL). Initial denaturation of 94ºCx5 min, was followed by18 cycles of 94ºCx20secs, 60ºCx30secs, 72ºCx30secs, with a final extension/elongation step of 72ºCx5min. PCR product was cleaned by the use of SPRI beads as per manufactor’s recommendation [Beckman Coulter, Indianapolis, IN]. Final product was resuspended in 20 ul of TrisEDTA. Final yields were quantified in a Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY) and quality of the library was assessed by running on a DNA1000 Bioanalyzer chip (Agilent Technologies, Santa Clara, CA). Libraries were normalized to 2nM and loaded on an Illumina HiSeq 2500 at 6pM, per manufacturer’s recommended protocol for 50bp single-read runs.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Targets_lane3.tar
Targets_lane34.tar
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| Data processing |
Basecalling performed using Illumina CASAVA v1.8.2.
ChIP-seq reads aligned to hg19 genome assembly using ELANDv2e from the Illumina CASAVA v1.8.2.
Peak calling performed using ChIPseeqer with default parameters.
Genome_build: hg19
Supplementary_files_format_and_content: targets files, the peak files generated by ChIPseeqer, are tab delimited with columns representing: chromosome, start_position, end_position, avg_p-value, score, posmaxpeakheight, maxpeakheight, relposmaxpeakheight(%), peak_size, mid_point
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| Submission date |
Dec 24, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Thadeous James Kacmarczyk |
| E-mail(s) |
thk2008@med.cornell.edu
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| Organization name |
Weill Cornell Medicine
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| Department |
Medicine/Hematology-Oncology
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| Lab |
Epigenomics Core
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| Street address |
1300 York Ave
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| City |
new york |
| State/province |
ny |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE64504 |
Multiplexing of ChIP-seq samples for a model experimental condition has minimal impact on peak detection |
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| Relations |
| BioSample |
SAMN03273126 |
| SRA |
SRX823962 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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