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| Status |
Public on Jan 15, 2022 |
| Title |
UBTF_0hrs_rep2_ChIP-Seq |
| Sample type |
SRA |
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| Source name |
ER-HoxA9 cell line with FKBPV-FLAG integrated into CEBPA locus
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| Organism |
Mus musculus |
| Characteristics |
agent: DMSO
time point: 0 hrs
chip antibody: Santa Cruz Biotechnology, #sc-13125
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| Treatment protocol |
For all experiments involving dTAGV-1 treatment, cells were seeded at a density of 2 x 10^5 cells/ml, and maintained below a maximum concentration of 1 x 10^6 cells/ml. Based on the requirements of individual experiments, 500 nM dTAGV-1 or DMSO were added to media at appropriate time points prior to cell harvest. For timecourse experiments, ‘0 hr’ samples was treated with DMSO for a period of time equal to the maximum duration of dTagV-1 treatment in that experiment.
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| Growth protocol |
ER-HoxA9 cell line was cultured at 37 °C in a 5 % CO2 atmosphere using RPMI-1640 media, supplemented with 10 % fetal bovine serum, 2 % stem cell factor [SCF] conditioned media (prepared from a Chinese hamster ovary cell line that stably secretes SCF), 0.5 µM β-Estradiol, and Penicillin/Streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
25 x 10^6 cells were crosslinked in 1% formaldehyde for 15 min at RT, and crosslinking reaction was quenched with glycine. To rupture cell and nuclear membranes, pelleted cells were snap-frozen in liquid nitrogen (30 sec), followed by thawing in 37 °C (1 min) for a total of three cycles, following which the cell pellet was stored at -80 °C. On the day of ChIP-Seq, cells were lysed in buffer containing MNase for chromatin fragmentation. MNase reaction was quenched by EGTA, and lysate was further sheared using a Bioruptor machine with two cycles of 30 sec ON and 30 sec OFF. Sheared extract was clarified by centrifugation, and to the clarified extract, 5 µl (50 ng) of Drosophila chromatin from S2 cells (Active Motif, #53083) was added as spike-in control. To ensure quantitative accuracy of spike-in, exactly 25 x 10^6 starting cells were used for each sample, and comparative samples were harvested and processed together. Extract was precleared by incubating overnight with mouse polyclonal IgG. The next morning, extract was clarified, and IgG was removed with Dynabeads. To the resultant supernatant (ChIP Input Lysate), 10 µg of the desired ChIP antibody was added, along with 2 µg of anti-H2Av (Active Motif, #61686) [which recognizes Drosophila-specific histone variant H2Av in spike-in chromatin], and incubated overnight at 4 °C with rotation. Lysate was clarified the next morning, and antibody-bound chromatin was pulled down with Dynabeads. Chromatin was eluted from Dynabeads, and treated with high salt, RNase A and Proteinase K. DNA was recovered using a Qiagen PCR purification kit, and eluted in 50 µl of 1X TE buffer.
All of the eluted DNA was used to construct NextSseq DNA libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Libraries were multiplexed using NEBNext Multiplex Oligos for Illumina, quantified using KAPA Quantification Kit, and sequenced on an Illumina Nextseq 500 platform.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
CEBPA Degron ER-HoxA9 line, treated with DMSO (0hrs) - UBTF ChIP-Seq Replicate 2
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| Data processing |
FASTQs were trimmed using Trimmomatic with the following parameters: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:30
Reads were mapped to a custom mm9_rDNA genome using Bowtie2 with the following parameter: -X 2000. This genome is the mm9 genome with a single 45,306 nt mouse rDNA repeat unit sequence (NCBI BK000964.3) inserted into chr12:2501-47806.
SAM files from Bowtie2 were converted to BAM using Samtools View with the following parameters: -F 4 -q 0. BAM files were sorted using Samtools Sort, and indexed using Samtools Index. Picard MarkDuplicates tool was used to remove PCR duplicate reads.
The IGVtools count feature was used to extract density across the rDNA sequence (chr12:2501-47806) from the BAM files.
Each FASTQ was also mapped in parallel to a Drosophila dm3 genome to quantify number of Drosophila reads pulled down by anti-H2av antibody. This read number was used to normalized rDNA mapping signal in each sample.
Genome_build: Custom-made mm9 genome with a single 45,306 nt mouse rDNA repeat unit sequence (NCBI BK000964.3) inserted into chr12:2501-47806
Supplementary_files_format_and_content: Excel spreadsheets showing normalized ChIP-Seq mapping signals across mouse rDNA sequence
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| Submission date |
Jan 12, 2022 |
| Last update date |
Jan 15, 2022 |
| Contact name |
Vikram R Paralkar |
| E-mail(s) |
vikram.paralkar@pennmedicine.upenn.edu
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| Phone |
215-573-9252
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| Organization name |
University of Pennsylvania Perelman School of Medicine
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| Department |
Division of Hematology and Oncology, Department of Medicine
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| Lab |
Paralkar Lab
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| Street address |
421 Curie Boulevard
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE193569 |
Assessing effect of CEBPA degradation on rRNA transcription machinery |
| GSE193651 |
Hematopoietic Transcription Factors Bind rDNA and Regulate rRNA Transcription |
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| Relations |
| BioSample |
SAMN24903198 |
| SRA |
SRX13760692 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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