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| Status |
Public on Jul 12, 2022 |
| Title |
P38i_IgG_3 |
| Sample type |
SRA |
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| Source name |
MDA-MB-231
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| Organism |
Homo sapiens |
| Characteristics |
chip antibody: IgG (Diagenode RIG001)
cell line: MDA-MB-231
cell type: triple-negative breast cancer (TNBC) cell
treatment: P38i
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| Growth protocol |
MDAMB231 cells were growth in 10% fetal bovine serum, 2mM glutamine, 1x penicillin/streptomycin. Cells were cultured at 37C at 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde, quenched with glycine, and frozen as a pellet. A total of 1 million cells per condition was used. We used the Diagenode ChIPmentation kit with dual indices, and followed the manufacturer’s protocol. Briefly, the cells were lysed and resuspended in 130ul of chromatin shearing. Shearing was done in a Covaris S2 instrument. A 15ul aliquot of the sheared chromatin was taken for assessment of the shearing size. The remaining sheared chromatin split for immunoprecipitation in the presence of H3K27me3 antibody (purchased Cell Signaling, provided by the investigator) and IgG as input. Four ug of each antibody was used per immunoprecipitation. Immunoprecipitation was done overnight at 4C with rotation. The immunoprecipitates were next washed before on-bead tagmentation at 37C for 10 minutes with agitation. The reaction was stopped by putting the samples on ice and additional washes, before processing for stripping, end filling and reverse crosslinking. qPCR was used to determine the optimal cycle number for enrichment PCR, according to the manufacturer’s protocol. The final amplified libraries were cleaned using AMPure XP beads at a 1.8:1 bead:DNA ratio, quantitated with Qubit HS dsDNA, and fragment size assessed on a TapeStation HS D1000 kit. The libraries were qPCR quantitated for pooling using the KAPA Illumina qPCR quantitation kit, and sequenced PE-50 on two NextSeq2000 P2 flow cells at the UM Advanced Genomics Core.
ChIPmentation
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 2000 |
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| Data processing |
We use FastQC (FastQC) (v0.11.8) to assess the overall quality of each sequenced sample.
We use TrimGalore (TrimGalore) (v0.4.5) and cutadapt (Martin) (v1.15) with the following parameters: –nextera -e 0.1 –stringency 6 –length 20 –nextseq 20.
We align trimmed reads to hg38 with Bowtie2 (Langmead and Salzberg, 2012) (v2.3.4.1) using default parameters with the exception of the following flags: -X 2000.
Duplicate reads are marked with Picard (Picard) (v2.20.2).
Alignments are filtered with samtools (Li et al., 2009) (v1.2) using the flags: -F4 -F8 -f3 -q10 -F1024.
Alignments completely overlapping blacklisted regions (ENCODE Blacklist Regions) are removed with bedtools (Quinlan and Hall, 2010) (v2.28.0).
Sample-wise peaks are called with epic2 (Stovner and Sætrom, 2019), a re-implementation of SICER (Zang et al., 2009), with flags: –false-discovery-rate-cutoff 0.05 –effective-genome-fraction 0.95 –guess-bampe.
Assembly: hg38
Supplementary files format and content: bigWig
Supplementary files format and content: BED (does not apply to IgG samples)
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| Submission date |
Jul 08, 2022 |
| Last update date |
Jul 14, 2022 |
| Contact name |
Bioinformatics Core |
| E-mail(s) |
bioinformatics@umich.edu
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| Organization name |
University of Michigan
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| Street address |
BRCF Bioinformatics Core, 2800 Plymouth Rd
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| City |
Ann Arbor |
| State/province |
Mi |
| ZIP/Postal code |
48109-2800 |
| Country |
USA |
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| Platform ID |
GPL30173 |
| Series (2) |
| GSE207794 |
EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression [ChIP-seq] |
| GSE207796 |
EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression |
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| Relations |
| BioSample |
SAMN29613321 |
| SRA |
SRX16101754 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6318394_P38i_IgG_3.bw |
79.5 Mb |
(ftp)(http) |
BW |
| GSM6318394_P38i_IgG_3.bw |
79.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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