 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 01, 2014 |
| Title |
ChIP-seq analysis of H3K27me3 in human monocytes P1 H3K27me3 |
| Sample type |
SRA |
| |
|
| Source name |
CD14++ CD16- monocytes from blood donor 1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: CD14++ CD16- monocytes
chip antibody: H3K27me3 Abcam ab6002
donor id: P1
disease state: control participant
|
| Treatment protocol |
n.a.
|
| Growth protocol |
n.a.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
A blood sample of 30 ml was taken and peripheral blood mononuclear cells were isolated using ficoll-based density gradient centrifugation. Further isolation of CD14++ CD16- monocytes was conducted using magnetic cell sorting to deplete CD16++ monocytes and separate CD14++ monocytes. Purity of isolation was checked by flow cytometric analysis of the CD14+ cell fraction. Cells were cross-linked for 10 min using formaldehyde (final concentration 1%). Fixation was quenched by addition of glycine to a final concentration of 125 mM. After washing, cells were resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris/HCl, pH8.1, 1x concentrated protease inhibitors) and sonciated using a Branson-250 Sonicator (http.www.bransonultrasonics.com) using the microtip setting. Fragment size of the obtained chromatin was checked to be between 100 bp and 300 bp. Sheared chromatin was diluted 1/10 with dilution buffer (SDS 0.01%, Triton X-100 1%, EDTA 1.2 mM, Tris/HCl, pH 8.1 16.7 mM, NaCl 167 mM). Chromatin was precleared using a protein A/G-sepharose mixture for 1 hr at 4°C and incubated with apprpriate antibodies over night. Immune complexes were precipitated with protein A/G-sepharose and washed with low salt (SDS 0.1%, Triton X-100 1%, EDTA 2 mM, Tris/HCl, pH8.1 20 mM, NaCl 150 mM), high salt (SDS 0.1%, Triton X-100 1%, EDTA 2 mM, Tris/HCl, pH8.1 20 mM, NaCl 500 mM), LiCl buffer (LiCl 0.25 M, NP40 1%, Deoxycholat 1%, EDTA 1 mM, Tris/HCl, pH8.1 10 mM) and twice with TE. After crosslink reversal, immunprecipitated nucleic acids were purified on GFX columns (GE Healthcare).
ChIP-seq library preparation using MicroPlex kit from Diagenode
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1000 |
| |
|
| Description |
ChIP-seq
|
| Data processing |
Quality control of FASTQ files was done using FastQC.
Removal of low quality reads was done using fastx_toolkit-0.06
Reads were aligned to a pre-compiled index of the hg19 genome using Bowtie (options: -k 1 -m 1) 0.12.5.
Duplicate reads were removed with Samtools' rmdup ( version 0.1.18) function.
Samtools was used to convert aligned reads to BAM format.
Genome_build: hg19
Supplementary_files_format_and_content: Smoothed and normalized coverage vectors were produced in 25 bp steps using custom software. Data was subsequently converted into indexed binary wiggle (bigwig) files using wigToBigWig utility downloaded from UCSC genome browser homepage.
|
| |
|
| Submission date |
Feb 03, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Marek Bartkuhn |
| E-mail(s) |
marek.bartkuhn@gen.bio.uni-giessen.de
|
| Organization name |
Justus-Liebig-University Giessen
|
| Department |
Biomedical Informatics and Systems Medicine
|
| Street address |
Aulweg 132
|
| City |
Giessen |
| State/province |
Hessen |
| ZIP/Postal code |
35392 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15433 |
| Series (1) |
| GSE54634 |
ChIP-seq mapping of histone modifications in CD14++ CD16- monocytes from human blood samples |
|
| Relations |
| BioSample |
SAMN02615839 |
| SRA |
SRX461539 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1320312_P1_H3K27me3_hg19_smoothed_normalized.bw |
265.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
