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| Status |
Public on Apr 10, 2024 |
| Title |
NALM6_polII_rep2 |
| Sample type |
SRA |
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| Source name |
NALM6
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| Organism |
Homo sapiens |
| Characteristics |
cell line: NALM6
cell type: B_ALL
read length: 50bp
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| Growth protocol |
RPMI 1640 + 10% serum
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA polymerase II ChIP-seq data were generated by first fixing 20 million Nalm6 cells in 1% formaldehyde (diluted from sigma F87750) at room temp for 10 minutes. Crosslinking was stopped with the addition of 2.5M glycine to a concentration of 0.125M, and the cells were then washed in ice-cold PBS. 5mg anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] (ab5408, lot: GR3264797-1) was prebound to 200ul of protein G dynabeads (invitrogen 10003D) overnight in 0.5% BSA in PBS. Fixed cell pellets (20M cells) were suspended in 1ml Farnham lysis buffer (5mM PIPES pH 8, 85mM KCl, 0.5% NP40, 1x protease inhibitors (Roche 11836170001)) and passed through a 18G needle 10x. Nuclei were pelleted and resuspended in 275ul of RIPA buffer (1x PBS, 1% NP40, 0.5% Sodium Deoxycholate, 0.1% SDS, 1x protease inhibitors) and sonicated on high power in 1.5ml tubes for 25 minutes (30s on/ 30s off) using a Diagenode Bioruptor Plus. 5% Input samples were taken from sonicated material and the remaining sonicated material was added to the pre-bound antibody/protein G beads to rotate overnight at 4C. The next day the supernatant was discarded, and the beads were washed 5x with ice cold LiCl buffer (100mM Tris pH 7.5, 500mM LiCL, 1% NP40, 1% sodium deoxycholate) and 1x with ice cold TE buffer (10mM Tris pH 7.5, 1mM EDTA). Samples were eluted from the washed beads using room temperature IP elution buffer (1% SDS, 0.1 M NaHCO3) at 65C for 1hr, vortexing every 15 minutes. The elution was then incubated at 65C overnight to reverse crosslinks. The next day DNA was purified using the QIAquick PCR purification kit (Qiagen 28104). DNA quantification was performed using the PicoGreen assay (Molecular Probes, Eugene, OR, P-7581). Sequencing libraries were generated from ChIP and input DNA by using the KAPA Hyper Prep kit (Roche, Basel, Switzerland, # 7962363001) according to the included manufacturer’s specifications, and quality was determined by using the Agilent TapeStation with D1000 screentape. Then, >50M 50-bp paired-end reads per sample were generated on the NovaSeq 6000.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Reads were quality checked using fastqc (v0.11.5)
and trimmed using trimgalore (v0.4.4)
before being mapped to the hg19 reference genome using bowtie2 (v2.2.9).
Sam files were converted to bam format using samtools (v1.2),
which were sorted using picard (v1.141).
Duplicates were removed using picard
and mitochondrial reads were removed using samtools.
For visualization, bam files from replicates were merged using samtools
and converted to bigwig format using deeptools (v3.5.0).
For peak calling, we used macs2 (v2.1.1), and only considered peaks called in both samples.
Assembly: HG19
Supplementary files format and content: .bigwig for coverage from merged replicates
Supplementary files format and content: .narrowpeak for PU.1 peaks called
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| Submission date |
Feb 09, 2023 |
| Last update date |
Apr 10, 2024 |
| Contact name |
Daniel Savic |
| E-mail(s) |
daniel.savic@stjude.org
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| Organization name |
St. Jude Children's Research Hospital
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| Department |
Pharmaceutical Sciences
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| Lab |
Savic
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| Street address |
262 Danny Thomas pl.
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| City |
Memphis |
| State/province |
TN |
| ZIP/Postal code |
38105 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE224204 |
Functional investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment |
| GSE224956 |
Functional investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment [ChIP-Seq] |
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| Relations |
| BioSample |
SAMN33227896 |
| SRA |
SRX19323308 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7036227_2049018_Nalm6_0hr_Rep2_PolII_ChIP_peaks.narrowPeak.gz |
3.3 Mb |
(ftp)(http) |
NARROWPEAK |
| GSM7036227_Nalm6_RNAP2_0hr_rep1_rep2_merged.bw |
156.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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