|
| Status |
Public on Mar 15, 2023 |
| Title |
Untagged_3-IAA_Rep2_6h |
| Sample type |
SRA |
| |
|
| Source name |
E14 mES cell line
|
| Organism |
Mus musculus |
| Characteristics |
cell line: E14 mES cell line
tag integration: osTIR1 was integrated into the genome using CRISPR-directed homologous recombination; separately, Supt16 was C-terminally tagged with the 37 kDa mini-AID tag and a triple V5 tag
treatment: Cells were left undisturbed on 10 cm plates for 48 hours prior to treatment. Cells were treated with 500 nM 3-IAA mixed into medium for the indicated time (hours). Cells were incubated at 37 degrees Celsius with 5% CO2 for the duration of treatment.
|
| Treatment protocol |
After 48 hours of undisturbed growth on 10 cm plates, medium was aspirated and cells were washed with DPBS. DPBS was aspirated and fresh medium was added containing 500 nM 3-Indoleacetic acid (3-IAA) or an equivalent volume of 100% ethanol. Plates were then left at 37 degrees Celsius with 5% CO2 for the indicated time before extraction.
|
| Growth protocol |
Murine embryonic stem cells were grown under feeder-free conditions in DMEM, supplemented with 10% Fetal Bovine Serum, 2 mM-Glutamine, 1X non-essential amino acids, 0.129mM 2-mercaptoethanol, 1000U/mL Leukemia Inhibitory Factor (LIF), 3 µM CHIR99021 GSK inhibitor (p212121), and 1 µM PD0325091 MEK inhibitor (p212121). Cells were passaged every 48 hours using trypsin (Gibco) and split at a ratio of ~1:8 with fresh medium. Routine anti-mycoplasma cleaning was conducted (LookOut DNA Erase spray, Sigma) and cell lines were screened by PCR to confirm no mycoplasma presence.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were trypsinized, quenched, pelleted, washed with cold PBS, and resuspended in hypotonic buffer. Nuclei were pelleted and lysed cell debris (supernatant) removed. Nuclei were flash-frozen until time of use.
Medium was aspirated and cells were washed with DPBS. DPBS was aspirated and cells were trypsinized. Cells were pelleted, washed with DPBS, and resuspended in hypotonic buffer. Nuclei were pelleted and lysed cell debris (supernatant) removed. Nuclei were flash-frozen until time of use. Nuclei were transposed for 30 minutes at 37C with 100 rpm shaking and Tn5 transposome (Diagenode). Libraries were amplified for 10 cycles of high-fidelity PCR
ATAC-seq
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
Transposed for 30 minutes at 37C with 100 rpm shaking and Tn5 transposome (Diagenode). Libraries were amplified for 10 cycles of high-fidelity PCR
|
| Data processing |
Reads were trimmed to 25 bp and barcode adapter sequences removed using trim_galore
Reads mapped to mm10 genome using bowtie2, allowing for 1 mismatch
Duplicates were removed using Picard
Reads were filtered for quality using SAMtools (MAPQ ≥ 10)
Reads were size-selected into nucleosome-free (1-100 bp) size class
Reads were processed in deepTools by using the bamCoverage command to generate bigwig files detailing coverage genome-wide in 1 bp bins with RPGC normalization, using an effectiveGenomeSize of 2308125349
Assembly: mm10
Supplementary files format and content: bigwig
|
| |
|
| Submission date |
Apr 28, 2022 |
| Last update date |
Mar 15, 2023 |
| Contact name |
David Charles Klein |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Pittsburgh
|
| Department |
Biological Sciences
|
| Lab |
Hainer Lab
|
| Street address |
4249 Fifth Ave
|
| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15213 |
| Country |
USA |
| |
|
| Platform ID |
GPL30172 |
| Series (2) |
| GSE181624 |
FACT maintains pluripotency factor expression through gene-distal regulation in embryonic stem cells |
| GSE201784 |
FACT maintains pluripotency factor expression through gene-distal regulation in embryonic stem cells [ATAC-Seq] |
|
| Relations |
| BioSample |
SAMN27928224 |
| SRA |
SRX15039653 |
