|Public on Sep 15, 2015
|diagnosis: Healthy control
tissue: Peripheral blood
cell type: CD4+CD45RO+ T cells
chip antibody: anti H3K27ac (ab4729; Abcam)
in vitro activation: yes
|To activate HC cells, HC PBMC were cultured for 16h with human T-activator CD3/CD28 dynabeads (1 cell: 3 beads)
|CD4+CD45RO+ cells were sorted from HC peripheral blood mononuclear cells (PBMC), either in vitro activated or not, and JIA PBMC and synovial fluid mononuclear cells (SFMC).
|Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo Research), end-repair, a-tailing, and ligation of sequence adaptors was done using Truseq nano DNA sample preparation kit (Illumina). Samples were PCR amplified, checked for the proper size range and for the absence of adaptor dimers on a 2% agarose gel and barcoded libraries were sequenced 75bp single-end on Illumina NextSeq500 sequencer.
|Illumina NextSeq 500
|Sample demultiplexing and read quality assessment was performed using BaseSpace (Illumina) software.
Reads were mapped to the reference genome (hg19) with Bowtie 2.1.0 using default settings.
SAM files were converted to BAM files using samtools version 0.1.19.
Peaks were called using MACS-2.1.0.
Enriched regions were identified compared to the input control using MACS2 callpeak --nomodel --exttsize 300 --gsize=hs -p 1e-9.
The mapped reads were extended by 300bp and converted to TDF files with igvtools-2.3.36
Differential binding analysis was performed using the R package DiffBind version 1.8.5. In DiffBind read normalization was performed using the TMM technique using reads mapped to peaks which were background subtracted using the input control.
BEDtools v2.17.0 was used for general manipulation of peak bed-files
Supplementary_files_format_and_content: Bigwig files
|Jul 31, 2015
|Last update date
|May 15, 2019
|Epigenetic profiling of Juvenile Idiopathic Arthritits (JIA) patients
|Epigenetic profiling and RNA-sequencing of Juvenile Idiopathic Arthritits (JIA) patients