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| Status |
Public on Jul 26, 2020 |
| Title |
ATF4 CHIP ATF4DN diff |
| Sample type |
SRA |
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| Source name |
erythroid progenitor cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: umbilical cord derived erythroid progenitor cells
cell line: HUDEP-2
antibody: rabbit anti-ATF4 (CST 11815S)
Stage: Differentiated
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
10 million cells per sample were harvested and cross-linked in 1% Formaldehyde. Cross-linking was quenched with the addition of 1.5M glycine. Samples were then lysed for 10 minutes at 4C in 50 mM Hepes–KOH, pH 7.5; 140 mM NaCl; 1 mM EDTA; 10% glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100. Cells were then centrifuged at 1500g for 3 minutes and the supernatant was discarded. The pellet was resuspended in 10 mM Tris–HCl, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA and incubated for 5 minutes at 4C. The cells were then centrifuged at 1500g for 3 minutes and the supernatant was discarded. The pellet was resuspended in 10 mM Tris–HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na–Deoxycholate; 0.5% N-lauroylsarcosine and sonicated using the Covaris S220 following manufacturer’s instructions. Protein A beads (ThermoFisher) were complexed with antibody and the antibody-bead complexes were incubated with cell lysates at 4C overnight with rotation. The antibodies used were rabbit anti-ATF4 (CST 11815S) and rabbit IgG (Novus Biologicals NBP2-24891). The beads were retrieved using a magnetic stand and rinsed with RIPA buffer. Elution buffer containing 50 mM Tris–HCl, pH 8; 10 mM EDTA; 1% SDS was added to the beads for reverse crosslinking at 65C overnight with shaking. After reverse crosslinking, the beads were removed. The eluted DNA was treated with RNaseA and Proteinase K and then purified using Qiagen MinElute PCR Purification Kit, following the manufacturer’s instructions.
Sequencing library was prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (E7647) and NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) following manufacturer’s instructions. Paired-end 150bp reads were generated on an Illumina NextSeq500 at the Functional Genomics Center Zürich (FGCZ) and demultiplexed.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
FastQC was used for initial quality control of reads. All samples, WT diff and undiff, HBBko diff and undiff, HSPC diff and undiff, ATF4ΔN diff, ATF4KO3, and IgG and HSPC IgG diff and undiff, were processed according to ENCODE guidelines for unreplicated transcription factor ChIP-seq analysis
raw reads were aligned against GRCh38 using bowtie2. Duplicate reads were marked using Picard’s MarkDuplicates and multimapping, low quality, duplicated and non-properly paired reads were removed. Library complexity measures and flagstats were generated for each BAM file.
BAM files were converted to tagAlign format and two subsampled pseudoreplicates were generated for each sample with half the total reads. Peak calling, fold change and p-value signal tracks were generated using MACS2
Irreproducible Discovery Rate (IDR) analysis was performed using self-pseudoreplicates and the main samples to obtain self-consistent sets of peaks. Final peak calls were filtered by ENCODE blacklist (Amemiya et al., 2019) and an IDR of 2% and a signal value > 30.
Sets of peaks for each comparison were analysed and associated to genes using the R package ChIPseeker (Yu et al., 2015) and Bioconductor hg38 TxDb (Team BC, Maintainer BP (2019). TxDb.Hsapiens.UCSC.hg38.knownGene: Annotation package for TxDb object(s). R package version 3.4.6.).
Genome_build: hg38
Supplementary_files_format_and_content: bigwig files
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| Submission date |
Jul 03, 2020 |
| Last update date |
Jul 27, 2020 |
| Contact name |
Jacob Corn Lab |
| Organization name |
ETH Zurich
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| Street address |
Otto-Stern-Weg 7
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| City |
ZURICH |
| State/province |
zURICH |
| ZIP/Postal code |
8093 |
| Country |
Switzerland |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE153767 |
ATF4 regulates MYB to increase g-globin in response to loss of β-globin [ChIP-seq] |
| GSE153768 |
ATF4 regulates MYB to increase g-globin in response to loss of β-globin |
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| Relations |
| BioSample |
SAMN15438806 |
| SRA |
SRX8664958 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4653925_ATF4DN_diff.bigwig |
289.3 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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