|
| Status |
Public on Mar 12, 2024 |
| Title |
cKIT+Sca1+Lin- cells, Murine Sf3b1 K700E, replicate 2, ATAC-seq |
| Sample type |
SRA |
| |
|
| Source name |
cKIT+Sca1+Lin- cells
|
| Organism |
Mus musculus |
| Characteristics |
cell line: cKIT+Sca1+Lin- cells
cell type: Hematopoietic progenitor cells
genotype: Sf3b1 K700E
|
| Treatment protocol |
Modified allele expression induced with doxycycline (1ug/ml) and maintained for 4 days (in 562 cells). For the murine model, SF3B1 mutant (and WT counterparts) were treated with tamoxifen daily x 5 days at 5 weeks of age. Mice were euthanized at end of week 8 and bone marrow was harvested to isolate the HSC progenitors.
|
| Growth protocol |
K562 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C. For the healthy volunteer and MDS patient samples, CD34 cells were isolated using FACS analysis. For the murine samples, cKit+Sca1+Lin- cells were isolated from bone marrow aspirates using FACS analysis
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
50,000 K562 cells or 5000 CD34+/murine HSC cells (flow sorted) were harvested, washed, and resuspended in PBS. Washed cells were resuspended in ATAC-seq resuspension buffer and centrifuged at 500g for 5 min in a 4 °C. Cell pellets were then resuspended in 50 μl of ATAC resuspension buffer. This cell lysis reaction was incubated on ice for 3 min. After lysis, 1 ml of RSB containing 0.1% Tween-20 (without NP-40 or digitonin) was added, and the tubes were inverted to mix. Nuclei were then centrifuged for 10 min at 500g at 4 °C. Supernatant was carefully removed and nuclei were resuspended in transposition mix containing transposase enzyme by pipetting up and down six times. Transposition reactions were incubated at 37 °C for 30 min in a thermomixer with shaking at 1,000 rpm.
DNA quality and size distribution were checked on Fragment Analyzer. 10 ng of DNA was used for library preparation according to NEBNext® Ultra II DNA Library Prep Kit
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Sequence reads were trimmed for adaptors with Cutadaptv4.1
Trimmed sequence reads were mapped to hg38 (for human samples) and mus musculus (for murine samples) using Bowtie2 aligner
To create promoter nucleosome occupancy plots, the locations of the mapped ATAC-seq reads were extended to 150 bp to represent sequenced fragments, and the resulting Bam files were used in the NucleoATAC page to generate output files in the bigWig file format. For ATAC peak analysis in K562 cell data, Epic2 package was used to call differentially enriched domains in WT and SF3B1K700E, using default parameters. Differentially ATAC-seq peaks were determined as those with a |log2 fold change(L2FC)| > 0.58 and adjusted p value < 0.05, calculated with DESeq2.
Assembly: hg38
Supplementary files format and content: bigWig
|
| |
|
| Submission date |
Feb 23, 2023 |
| Last update date |
Mar 12, 2024 |
| Contact name |
MANOJ PILLAI |
| E-mail(s) |
MANOJ.PILLAI@YALE.EDU
|
| Organization name |
YALE UNIVERSITY
|
| Lab |
PILLAI LAB
|
| Street address |
300 GEORGE STREET #786
|
| City |
NEW HAVEN |
| State/province |
CT |
| ZIP/Postal code |
06511 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE225993 |
Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [ATAC-seq] |
| GSE226003 |
Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape. |
|
| Relations |
| BioSample |
SAMN33427645 |
| SRA |
SRX19490161 |
