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| Status |
Public on Dec 31, 2018 |
| Title |
EZH2-null TFH, biological rep2 [ATAC-seq] |
| Sample type |
SRA |
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| Source name |
EZH2-null mice, LCMV Armstrong D8
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| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6 x 129SV
genotype/variation: EZH2-null
cell type: TFH
treatment: LCMV Armstrong infection
time point: Day 8
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATAC-seq:
50,000 sorted cells were treated with lysis buffer and then centrifuged. The nuclei preparation was harvested after discarding the supernatant.
Libraries were prepared with the Nextera DNA Sample Preparation Kit (FC-121-1030, Illumina). Briefly, the nuclei preparation was labeled with Nextera enzyme and immediately amplified by PCR of 9-10 cycles with barcoded primers and sequenced on NextSeq500 in a 150 bp/150 bp or 76 bp/76 bp paired end run.
ChIP-seq:
DNA-protein complexes of target cells were extracted and fixed with the Simple ChIP Enzymatic Chromatin IP Kit (Magnetic Beads) (#9003; Cell Signaling Technology) according to the manufacturer's instruction.
Chromatin fragments were immunoprecipitated with anti-H3K27me3 (Cell Signaling Technology, #9733, Clone number: C36B11) with ChIP Grade Protein G magnetic Beads (#9006; Cell Signaling Technology). The Illumina’s TruSeq indexed pair-ended DNA library preparation protocol was performed automatically on the SPRIworks system (Beckman Coulter). By using cartridge and method card specific to Illumina sequencing system, a fragment library can be prepared for Illumina sequencers. After individual libraries were constructed, qualities and band-sizes were assessed using Bioanalyzer High Sensitivity Chip (Agilent Technologies) and Qubit (Life Technologies). Libraries were also quantified by qPCR using the Library Quantification Kit for Illumina sequencing platforms (KAPA Biosystems), using an ABI 7900HT Real-Time PCR System (Life Technologies). Libraries were normalized to a working concentration of 10 nM, using the molarity calculated from qPCR and adjusted for fragment size with the Bioanalyzer analysis.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
KOTFH_Rep2
processed data file: KOTFH.final.bigwig
processed data: KO_WT_TFH.txt
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| Data processing |
ATAC-seq:
Raw sequencing reads were first trimmed for both adapters and quality and filtered for quality using Trim Galore! v0.4.4, which is a wrapper based on CutAdapt v1.14 and FastQC v0.11.5.
Paired-end reads which passed quality control (QC) were then aligned against mm10 using Bowtie2 v2.2.9.
The resultant BAM files were then filtered again to remove unmapped reads, mate-unmapped reads, non-primary aligned reads, reads that fail platform quality checks and PCR duplicate reads using SAMtools v1.4.1 ( -F 1804). In addition, reads mapped to ChrM were also removed and PCR duplicate reads were further marked up and removed using Picard v2.16.0 MarkDuplicates. The insert size distribution were then calculated using Picard v2.16.0 CollectInsertSizeMetrics.
Since Tn5 transposase binds as a dimer and insert two adaptors separated by 9bp, all aligned reads were shifted +4 bp on the positive strand and -5bp on the negative strand using deepTools v2.5.2 alignmentSieve.
Following that, peak calling was performed using MACS2 v2.1.1 with a q-value threshold of 0.01. Peaks that overlap with the Encode black list regions were then removed using bedtools v2.26.0 intersect. We then merged peaks from all replicates and filtered peaks that are not reproducible, using Irreproducible Discovery Rate (IDR) (IDR < 0.005), across at least one pair of replicates in each sample group. Subsequently, depending on the comparisons required for different purposes, filtered peaks from multiple sample groups were merged using bedtools v2.26.0 merge to create an atlas of genome wide accessible chromatin regions for further analysis.
For each analysis that compares across multiple sample groups, all required shifted BAM files of all replicates of those samples were used to generate an accessibility matrix by counting the normalized reads (by reads per million, RPM) within each peak region of the corresponding atlas peak file, using deepTools v.2.5.4 multiBamSummary in BED-mode. The resulting matrix was passed to DESeq2 v1.16.1 to calculate the differential accessibility of the peaks of the relevant pairs. PCA was then plotted using DESeq2 v1.16.1.
ChIP-seq:
Similar to the above ATAC-seq analysis, briefly, the raw sequencing reads from ChIP-seq were first trimmed and filtered with Trim Galore! v0.4.4, and then aligned in single-end mode and against mm10 using Bowtie2 v2.2.9.
The reads were then filtered using samtools to remove low quality reads and unmapped reads. Duplicate reads were also filtered using Picard v2.16.0 MarkDuplicates.
Peak calling was done by MACS2 v2.1.1 with a q-value threshold of 0.01 and the --SPMR flag. The resultant bedgraph files were used to build coverage plot using MACS2 v2.1.1 bdgcmp with a logLR method and a p value of 1e-5.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Bigwig: Coverage plots (Replicates FDR filtered and combined; 1 per sample group).
Supplementary_files_format_and_content: narrowPeak: Peak calling results from MACS2 (1 per sample).
Supplementary_files_format_and_content: txt: Raw counts matrix of reads per atlas peak (1 per analysis group).
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| Submission date |
Sep 26, 2018 |
| Last update date |
May 24, 2023 |
| Contact name |
Jun Huang |
| E-mail(s) |
[email protected]
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| Phone |
7737023218
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| Organization name |
University of Chicago
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| Department |
Institute for Molecular Engineering
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| Lab |
Huang Lab
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| Street address |
Eckhardt, 5640 S Ellis Ave
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE120532 |
Genome-wide maps of chromatin state in naïve CD4+ T cells, WT/EZH2-null TFH and TH1 cells |
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| Relations |
| BioSample |
SAMN10133764 |
| SRA |
SRX4742279 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3402634_KOTFH_Rep2.final.narrowPeak.gz |
618.7 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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