|
Status |
Public on Mar 12, 2024 |
Title |
Mouse cells, SF3B1 Sf3b1K700E, Ser2P CTD |
Sample type |
SRA |
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|
Source name |
n/a
|
Organism |
Mus musculus |
Characteristics |
cell line: n/a cell type: lineage -ve marrow cells genotype: Sf3b1K700E
|
Treatment protocol |
Modified allele expression induced with doxycycline (1ug/ml) and maintained for 4 days
|
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.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Formaldehyde was directly added to the media to a final concentration of 1% and 10 million cells were incubated for 15 min. Glycine was added to quench the reaction for 5 min. Cells were spun down and pellet was washed twice with PBS . The cell pellet was lysed on ice in cell lysis buffer, pelleted and resuspended in MNase digestion buffer containing MNase, followed by nuclear lysis in nuclear lysis buffer. Sonication was performed with a sonic Dismembranator.100 μg of chromatin was used for each IP. Either pan-Pol2 CTD, Ser5P CTD, or Ser2P CTD antibodies was mixed with diluted chromatin and subjected to IP performed on a rotating wheel at 4°C overnight. Pierce Protein A/G Magnetic beads were added to the chromatin-antibody mixture and 4°C rotation continued for 3 more hours. Beads were washed with wash buffers. Immuno-bound chromatin was eluted at 55°C for 1 hour with elution buffer and de-crosslinked overnight at 65°C. DNA was extracted with phenol:chloroform:isoamyl alcohol and precipitated for 60 min at −20°C with sodium acetate and ethanol. Pellet was washed with 70% ethanol and resuspended in TE buffer. 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. Purity and size distribution of the libraries were estimated using Fragment Analyzer.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 2500 |
|
|
Data processing |
Sequence reads were trimmed for adaptors with Cutadaptv4.1 Trimmed sequence reads were mapped to hg38 using Bowtie2 aligners The respective IP and Input BAM files were compared to each other while being simultaneously normalized for sequencing depth (using RPKM), and reformatting to the bigWig file format using deepTools BamCompare program. Assembly: hg38 Supplementary files format and content: bigWig
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Submission date |
Jan 31, 2024 |
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) |
GSE226003 |
Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape. |
GSE254792 |
Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [Murine ChIP-Seq] |
|
Relations |
BioSample |
SAMN39710322 |
SRA |
SRX23484915 |