|
| Status |
Public on Nov 22, 2019 |
| Title |
H3K27ac_ATrep2 |
| Sample type |
SRA |
| |
|
| Source name |
Human PBMCs
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Human PBMCs
age: 70 years
Sex: Male
sequencing adapter: TGACCA
cell type: adult CD4+CD25hiCD127lo regulatory T
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were sorted by FACS, and DNA was extracted from lysed cells following conventional ATACseq and ChIPseq protocols
ATACseq libraries were constructed using the standard Illumina Nextera protocol. H3K27ac ChIPseq libraries were constructed using a standard Illumina protocol using Truseq adapters.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Fastq files were mapped to the genome using Bowtie 2 with the following parameters: -p 10 -q -D 15 -R 10 -L 22 -i S,1,1.15
Mapped reads were filtered to keep uniquely mapping, non-duplicated reads using Samtools. Reads with q>30 were kept.
MACS2 was used for peak calling for ChIPseq dataset using the following settings: callpeak -t ${K27ac_SAMPLE} -c ${INPUT} -f BAM -g hs -q 0.01
The ROSE algorithm was used to define superenhancers. Briefly, each peak file for each ChIPseq replicate was converted into GFF format and peaks within 2kb of the TSS were removed. Superenhancer calling was performed with the following settings: -g hg19 -i ${GFF} -r ${K27acBAM} -c ${INPUTBAM} -s 12500 -o ${OUTPUT}
Generation of read count tables across a masterlist of all superenhancers/transcriptional enhancers for DESeq2 analysis. Enhancers across all replicates were merged using bedops -merge to create a masterlist of SEs and Tes shared across all cell types and cell origin. Bedtools -multicov was utilized to map reads from ATACseq and ChIPseq bam files onto enhancer regions.
Bigwig files for ATACseq were generated by first calculating tag densities as the number of tags that map to within 75 bp of each 20bp genomic bin. Tag insertion positions were calculated as +4bp from the upstream end of + strand tags and -5 bp from - strand tags. Blacklist regions were also removed using known regions from the ENCODE consortium. Regions with tag density greater than 40 were identified as accessible regions.
Bigwig files for ChIPseq were generated by first calculating tag density. The midpoint of each fragment size for each sample replicate was calculated and tags were mapped to within 75 bp of each 20bp genomic bin for both input and immunoprecipitated samples. Blacklist regions were also removed using known regions from the ENCODE consortium. Tag counts were normalized to library size, and input counts were subtracted before conversion into the bigwig format
Genome_build: hg19
Supplementary_files_format_and_content: BED file of all merged superenhancers/transcriptional enhancers. Bigwig files for each individual biological replicate for ATACseq and H3K27c ChIPseq
|
| |
|
| Submission date |
Feb 12, 2018 |
| Last update date |
Nov 24, 2019 |
| Contact name |
Melissa Ng |
| E-mail(s) |
[email protected]
|
| Organization name |
University of California, San Francisco
|
| Lab |
Burt Lab
|
| Street address |
35 Medical Centre Way, Regeneration Medicine Building 903B
|
| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94143 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE110468 |
Helios enhances the preferential differentiation of human fetal CD4+ naïve T cells into regulatory T cells. [Epigenetics] |
| GSE110472 |
Helios enhances the preferential differentiation of human fetal CD4+ naïve T cells into regulatory T cells. |
|
| Relations |
| BioSample |
SAMN08518272 |
| SRA |
SRX3680716 |
