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| Status |
Public on Feb 19, 2021 |
| Title |
Input_iPSC |
| Sample type |
SRA |
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| Source name |
induced pluripotent stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: CHD6 wild type
passages: 20-25
chip antibody: none
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| Growth protocol |
iPSCs were cultured in FTDA media
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each batch of ChIP experiments ~12 million cells were crosslinked in 2% PFA for 45 min at 4°C. From this point onward, cells were processed via the ChIP-IT High Sensitivity kit (Active motif) as per manufacturer’s instructions, but using the NEXSON protocol for the isolation of nuclei [1]. Chromatin was sheared to 200-500 bp fragments on a Bioruptor Plus (Diagenode; 2x 20-26 cycles of 30s “on” and 30s “off”, at the highest power setting), and immunoprecipitations were carried out by adding 4 μg of the appropriate antiserum (CHD6, A301-221A Bethyl; TF3B, A303-673A Bethyl) to ~30 μg of chromatin and incubating on a rotator overnight at 4°C in the presence of protease inhibitors. Following addition of protein A/G agarose beads and washing, DNA was purified using the ChIP DNA Clean & Concentrator kit (Zymo Research) and used in qPCR or sequencing on a HiSeq4000 platform (Illumina).
For each batch of ChIP experiments ~12 million cells were crosslinked in 2% PFA for 45 min at 4°C. From this point onward, cells were processed via the ChIP-IT High Sensitivity kit (Active motif) as per manufacturer’s instructions, but using the NEXSON protocol for the isolation of nuclei [1]. Chromatin was sheared to 200-500 bp fragments on a Bioruptor Plus (Diagenode; 2x 20-26 cycles of 30s “on” and 30s “off”, at the highest power setting), and immunoprecipitations were carried out by adding 4 μg of the appropriate antiserum (CHD6, A301-221A Bethyl; TF3B, A303-673A Bethyl) to ~30 μg of chromatin and incubating on a rotator overnight at 4°C in the presence of protease inhibitors. Following addition of protein A/G agarose beads and washing, DNA was purified using the ChIP DNA Clean & Concentrator kit (Zymo Research) and used in qPCR or sequencing on a HiSeq4000 platform (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
ChIP-seq reads were aligned to the hg19 using BWA, reads with mapping quality below 10 were filtered out, duplicates were removed using Picard.
peaks were called using MACS2 peak caller with the following arguments: -f BAM -g hs -s 100 --keep-dup 1 -q 0.01 (for CHD6) and -q 0.005 (for TFEB)
Genome_build: hg19
Supplementary_files_format_and_content: The Wig file is generated from the bedGraph file, using an in-house script that computes the extended read coverage at a user-defined step size (default: 20 bp). The extended read coverage is normalized to a library size of one million mapped reads, and converted into the TDF format using the toTDF command from the igvtools package (http://www.broadinstitute.org/software/igv/igvtools).
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| Submission date |
Aug 20, 2019 |
| Last update date |
Feb 19, 2021 |
| Contact name |
Yulia Kargapolova |
| Organization name |
Cologne University
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| Department |
CMMC
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| Lab |
AG Papantonis
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| Street address |
Robert-Koch-Straße 21
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| City |
Cologne |
| ZIP/Postal code |
50931 |
| Country |
Germany |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE136057 |
Impaired CHD6 function links misregulation of autophagy and DNA damage response to premature ageing [ChIP-Seq] |
| GSE161120 |
Impaired CHD6 function links misregulation of autophagy and DNA damage response to premature ageing |
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| Relations |
| BioSample |
SAMN12608730 |
| SRA |
SRX6743816 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4040265_K002000297_98787_S30_L005_R1_001.SE.U1.dedup.s1.bam.s20_total_based_norm.tdf |
450.4 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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