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| Status |
Public on Jul 09, 2021 |
| Title |
µATACseq: ATACseq with as few as 100 cells |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
μATACseq data generated from 100, 500 and 1,000 FACS-purified Atoh1-GFP expressing cerebellar granule precursors (CGPs) are consistent with those from 20,000 CGPs by the standard ATACseq protocol (R > 0.85), with comparable percentages of both the reads mapped to the promoters (±1000bp Transcription Start Sites), and to the distal elements. Of the peaks identified from the standard ATACseq method, more than 70% are detected among those from the μATACseq with as low as 100 cells. In addition, μATACseq lowers the sequencing cost by generating 10-fold fewer mitochondrial reads and up to 1.7-fold fewer PCR duplicates. Surprisingly, the μATACseq protocol is robust over a 10-fold difference in the transposase-to-cell ratios measured by (1) the fragment size distribution inferred from pair-end sequencing, (2) the depth of the predicted transcription factor footprints, (3) and the concordance between biological replicates. Further more, μATACseq is fast and convenient, does not require multiple washing steps or nuclear isolation, and demands only 20 minutes prior to DNA purification and sequencing library amplification.
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| Overall design |
µATACseq was validated using CGPs since they are aboudant and they express ATOH1-GFP. FACS-purified cells (100, 500,1000 cells per reaction) were centrifuged to remove the supernatant. 20 μL lysis buffer (10 mM Tris HCl (pH 7.4), 5 mM MgCl2, 10% DMF, 0.2% N-P40) was added, followed by pipetting 6-10 times to release the nuclei. 30 μL reaction buffer (10 mM Tris HCl (pH 7.4), 5 mM MgCl2, 10% DMF, 1000 unit transposome) was added directly to the cell lysis, and mixed by vortexing. The reaction was incubated at 37 °C for 20 minutes. 350 μL ERC buffer (MiniElute, Qiagen) was added to stop the reaction. After DNA purification (MiniElute, Qiagen), ATACseq libraries were constructed as previously described (Buenrostro et al., 2013).
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| Contributor(s) |
Haoze Y, Neil S |
| Citation(s) |
34266958 |
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| Submission date |
May 11, 2020 |
| Last update date |
Oct 08, 2021 |
| Contact name |
Neil Segil |
| E-mail(s) |
nsegil@med.usc.edu
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| Phone |
3234421549
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| Organization name |
University of Southern California
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| Department |
BCC
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| Lab |
503K
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| Street address |
1425 san pablo st, BCC 503K
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90033 |
| Country |
USA |
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| Platforms (1) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
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| Samples (8)
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| This SubSeries is part of SuperSeries: |
| GSE150391 |
POU4F3 Pioneer Activity Enables ATOH1 to Drive Diverse Mechanoreceptor Differentiation Through a Feed-Forward Epigenetic Mechanism |
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| Relations |
| BioProject |
PRJNA631703 |
| SRA |
SRP261102 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE150275_RAW.tar |
23.5 Mb |
(http)(custom) |
TAR (of NARROWPEAK) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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