|
| Status |
Public on Oct 14, 2020 |
| Title |
Effects of sheared chromatin length on ChIP-seq quality and sensitivity |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
Third-party reanalysis
|
| Summary |
Chromatin immunoprecipitation followed by massively parallel, high throughput sequencing (ChIP-seq) is the method of choice for identifying, on a genome-wide scale, the segments of DNA bound by specific transcription factors (TFs) or in chromatin with particular histone modifications. However, the quality of ChIP-seq datasets vary over a wide range, with a substantial fraction being of intermediate to poor quality. Such experimental variability can lead to many false positives or false negatives, impairing the ability to interpret the data. Thus, it is important to discern and control the factors that contribute to variation in ChIP-seq. In this study we focus on the sonication step to produce sheared chromatin, a variable controllable by the user and applicable to all ChIP-seq protocols. We systematically varied the amount of shearing of fixed chromatin from a mouse erythroid cell line, carefully measured the distribution of resultant fragment lengths using the Agilent Bioanalyzer 2100, and then immunoprecipitated these batches of chromatin using highly specific antibodies against either TAL1 or CTCF. We found that the level of sonication, which was affected by both the number of sonication cycles, as well as the starting cell number, had a pronounced impact on the quality of resulting ChIP-seq signals. Specifically, over-sonication led to degradation of quality (e.g. increased background and reduction in signal), while the impact of under-sonication of chromatin differed between the two transcription factors, leading to the loss of sites occupied by TAL1 but not those bound by CTCF. We leveraged these findings to produce a set of CTCF ChIPs-seq datasets in primary hematopoietic progenitor cells, including several rare cell types. Together, these results suggest that the amount of sonication is a key variable in success of ChIP-seq experiments, and that carefully monitoring the level of chromatin sonication is one way to improve ChIP-seq quality and reproducibility, which in turn facilitates low input ChIP-seq in rare cell types.
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| |
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| Overall design |
We systematically varied the extent of sonication of fixed chromatin from G1E-ER4+E2 cells, carefully measured the distribution of resultant fragment lengths using the Agilent Bioanalyzer 2100, and then immunoprecipitated these batches of chromatin using highly specific antibodies against either TAL1 or CTCF. Following ChIP-seq library preparation, these samples were sequenced on the Illumina NextSeq 500. The datasets were then examined by both subjective visual assessments of the signal tracks at various loci on the genome browser, as well as established, genome-wide, objective quality metrics to determine the effects of sheared chromatin length on ChIP-seq quality and sensitivity. These findings were then leveraged to produce a set of CTCF ChIP-seq datasets in 8 bone marrow-derived, hematopoietic stem and progenitor cells, including several rare cell types.
Samples GSM946520 (GSE36028, GSE49847, GSE51338) and GSM995439 (GSE36029, GSE49847, GSE51338) were used as the input samples for the G1E-ER4+E2 samples.
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| |
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| Contributor(s) |
Keller CA, Wixom AQ, Heuston EF, Giardine BM, Hsiung CC, Long MR, Miller A, Wixom AQ, Anderson SM, Cockburn AK, Blobel GA, Bodine DM, Hardison RC |
| Citation(s) |
33788948 |
| Submission date |
Oct 13, 2020 |
| Last update date |
Aug 24, 2021 |
| Contact name |
Ross Hardison |
| E-mail(s) |
rch8@psu.edu
|
| Organization name |
Pennsylvania State University
|
| Street address |
303 Wartik Lab
|
| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
| |
|
| Platforms (3) |
| GPL13112 |
Illumina HiSeq 2000 (Mus musculus) |
| GPL16417 |
Illumina MiSeq (Mus musculus) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
|
| Samples (65)
|
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| Relations |
| Reanalysis of |
GSM946520 |
| Reanalysis of |
GSM995439 |
| BioProject |
PRJNA669079 |
| SRA |
SRP287324 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE159503_1295.mm10.broadpeak.gz |
282.9 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_1908.mm10.broadpeak.gz |
302.8 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_1909.mm10.broadpeak.gz |
130.3 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_1910.mm10.broadpeak.gz |
333.3 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_1926.mm10.broadpeak.gz |
105.3 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_1927.mm10.broadpeak.gz |
448.7 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_1976.mm10.broadpeak.gz |
385.7 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_1977.mm10.broadpeak.gz |
505.3 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_1978.mm10.broadpeak.gz |
210.3 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_1979.mm10.broadpeak.gz |
544.1 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_1980.mm10.broadpeak.gz |
189.7 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_335.mm10.broadpeak.gz |
253.7 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_366.mm10.broadpeak.gz |
89.8 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_369.mm10.broadpeak.gz |
295.9 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_485.mm10.broadpeak.gz |
50.3 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_505.mm10.broadpeak.gz |
5.2 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_580.mm10.broadpeak.gz |
159.0 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_581.mm10.broadpeak.gz |
7.4 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_582.mm10.broadpeak.gz |
3.0 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_583.mm10.broadpeak.gz |
46.4 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_584.mm10.broadpeak.gz |
23.1 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_585.mm10.broadpeak.gz |
17.7 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_670.mm10.broadpeak.gz |
4.2 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_671.mm10.broadpeak.gz |
40.1 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_672.mm10.broadpeak.gz |
65.2 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_673.mm10.broadpeak.gz |
115.4 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_674.mm10.broadpeak.gz |
133.3 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_692.mm10.broadpeak.gz |
202.4 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_693.mm10.broadpeak.gz |
233.5 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_694.mm10.broadpeak.gz |
207.6 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_695.mm10.broadpeak.gz |
286.0 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_696.mm10.broadpeak.gz |
186.5 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_702.mm10.broadpeak.gz |
65.3 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_703.mm10.broadpeak.gz |
57.5 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_704.mm10.broadpeak.gz |
51.3 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_705.mm10.broadpeak.gz |
24.8 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_706.mm10.broadpeak.gz |
8.4 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_707.mm10.broadpeak.gz |
39.0 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_732.mm10.broadpeak.gz |
208.8 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_733.mm10.broadpeak.gz |
157.4 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_734.mm10.broadpeak.gz |
169.1 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_735.mm10.broadpeak.gz |
171.4 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_736.mm10.broadpeak.gz |
94.5 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_785.mm10.broadpeak.gz |
211.4 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_786.mm10.broadpeak.gz |
92.7 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_787.mm10.broadpeak.gz |
172.0 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_788.mm10.broadpeak.gz |
39.1 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_789.mm10.broadpeak.gz |
178.1 Kb |
(ftp)(http) |
BROADPEAK |
| GSE159503_Keller_etal_2021_Supplemental_Tables_GEO.xlsx |
94.5 Kb |
(ftp)(http) |
XLSX |
| GSE159503_RAW.tar |
159.5 Gb |
(http)(custom) |
TAR (of BROADPEAK, BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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