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| Status |
Public on Aug 12, 2024 |
| Title |
HFK gH2AX 2 |
| Sample type |
SRA |
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| Source name |
Foreskin
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Foreskin
cell line: HFKs
cell type: Keratinocytes
chip antibody: Phospho-Histone H2A.X (Ser139) (D7T2V) Mouse mAb #80312
treatment: None
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| Growth protocol |
HFK and CIN 612 cells were maintained in E-media containing 10% FBS DMEM
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
1 × 107 cells were harvested and collected in Southern lysis buffer before being treated with RNase A (5 ng/mL) and Proteinase K (7.5 ng/mL) at 37 °C overnight. DNA was purified from these samples using phenol-chloroform extractions, and 25 to 50 mg of DNA was used for each sample. DNA was sheared using a Bioruptor (Diagenode) on high power, 30 s on/90 s off cycles for 20 min. Input DNA was removed before loading the samples into preblocked magnetic beads in IP buffer containing 2 ug of the H3K36me3 antibody. Immunoprecipitations were allowed to incubate overnight at 4 °C while rotating. The next day, samples were washed 8 times with RIPA buffer for 5 min while rotating. One wash in TE buffer was performed before samples were eluted for 10 min at 65 °C in 10% sodium dodecyl sulfate (SDS), 10 mM Tris pH 7.4, 50 mM ethylenediaminetetraacetic acid (EDTA). DNA was purified from these elutions using a PCR purification kit (Qiagen) and stored at −20 °C.
Samples were stored at -80° C until being shipped to Admera Biosciences (NJ) or Northwestern sequencing core (NU seq), who performed the sequencing experiments. Briefly, the library was prepared using a KAPA HyperPrep Kit (Kapa Biosystems) following the manufacturer’s recommendation. Input DNA was end-repaired and 3’-dA tailed. Adapter was then ligated to the DNA, and the ligated product was PCR amplified and cleaned up using the SPRIselect Reagent (Beckman Coulter). Quality control was then performed for the final library, followed by sequencing.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
FastQC (v0.11.8) was used to check the quality of raw and trimmed reads. Trimmomatic (v0.38) was used to cut adapters and trim low-quality bases with a default setting. BWA (v0.7.10-r789) was used to map the trimmed reads to the reference genome* using the Burrows-Wheeler Alignment algorithm (BWA-MEM). Mapped reads that have low-quality MAPQ score (MAPQ < 10), not-properly-paired, or duplicated (assessed with Picard tools (v 2.20.4)) were removed. BAM was used to generate BW format (normalized by RPKM) for visualization. MACS (v2.2.4) was chosen to call peaks.
Agreement between biological replicates was assessed using multiBAMSummary (Galaxy Version 3.5.4+galaxy0), and then plotting principal component analyses using plotPCA (Galaxy Version 3.5.4+galaxy0) and plotting Pearson coefficients as a heatmap using plotCorrelation (Galaxy Version 3.5.4+galaxy0) (Supp Fig 8). From the BAM files, BAM Compare (Galaxy Version 3.5.4+galaxy0) was used to normalize log2 IP to input ratios for both biological replicates of H3K36me3, H3K9me3, and gH2AX ChIPs from HFK and CIN 612 cells. ComputeMatrix was used to prepare files for visualization via plotProfile and plotHeatmap. CHIPseeker (Galaxy Version 1.28.3+galaxy0) was used on the BED files generated by Admera Biosciences or NUseq to determine the genomic distribution of each modified histone. MACS (v2.2.4) was used to call peaks. HOMER (Galaxy Version 4.11+galaxy0) was used to annotate where peaks occurred relative to their genomic location (intron, exon, etc.) and the corresponding gene name.
Assembly: Hg38
Supplementary files format and content: bigWig
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| Submission date |
Aug 06, 2024 |
| Last update date |
Aug 23, 2024 |
| Contact name |
Conor Templeton |
| E-mail(s) |
Conor.Templeton@northwestern.edu
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| Organization name |
Northwestern University
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| Street address |
300 E Superior Street
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| City |
Chicago |
| ZIP/Postal code |
60611 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE274119 |
HPV induced R-loop formation represses innate immune gene expression while activating DNA damage repair pathways [gH2AX ChIP seq] |
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| Relations |
| BioSample |
SAMN43053950 |
| SRA |
SRX25621636 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8443709_HFK_gH2AX_2.ucsc.bigWig |
22.7 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
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