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| Status |
Public on Oct 04, 2023 |
| Title |
iFoxL2, mES+24hrs, antiFLAG-ChIP-exo, rep2 |
| Sample type |
SRA |
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| Source name |
MC1(+iFoxL2)
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| Organism |
Mus musculus |
| Characteristics |
cell line: MC1(+iFoxL2)
cell type: embryonic stem cells
genotype: 129S6/SvEvTac
treatment: Dox removal +24hrs
chip-seq antibody: F1804 Sigma, lot SLBW5142
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| Treatment protocol |
To induce transgene TFs, Dox was removed by washing three times with PBS at 3 hr intervals and cells were grown in DMEM differentiation medium containing 10% FBS for the stated amount of time. Chromatin was crosslinked using 1% formaldehyde for 10 minutes.
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| Growth protocol |
Generation of the mES transgenic cell lines used in this study is described in [PMID: 19796622]. These cell lines were part of NIA (National Institute on Aging/NIH) mouse ES cell collection. Cell lines are derived from MC1 mouse ES cells (129S6/SvEvTac). Doxycycline-repressible expression constructs containing the relevant TF ORF, C-terminally tagged with His6-FLAG, and a puromycin resistance gene were inserted into the expression-competent Rosa26 locus. ES cells were cultured in a gelatin-coated dish with DMEM medium containing 15% FBS, 1000 U/ml LIF, 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, 2 mM glutamate, 0.1 mM ß-mercaptoethanol, 0.2µg/ml Doxycycline and 1.0μg/ml puromycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysis was performed in Farnham cell lysis buffer for 20mins at 4°C. At the 10-min mark, cells were pushed through a 25-gauge needle five times to enhance cellular lysis. Nuclei were then isolated by pelleting at 10,000 rpm for 3 min. Nuclei were resuspended in RIPA buffer (25 million cells to 1 mL of buffer) for an additional 20 min at 4°C and then pelleted again at 10,000 rpm for 3 min. Disrupted nuclei were then finally resuspended in 1× PBS (25 million cells to 1 mL of buffer) and sonicated for 10 cycles (30-on/30-off) in a Diagenode pico.
Solubilized chromatin was pre-cleared by centrifugation at maximum speed for 15 min at 4°C before incubating with 10 μL pAG Dynabeads, preloaded with 3 μg of antibody overnight, then sequentially processed through A-tailing, first adapter ligation, Phi29 fill-in, lambda exonuclease digestion, cross-link reversal, second adapter ligation, and PCR followed by size selection for final high-throughput sequencing.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Basecalls were performed using using bcl-convert verison 00.000.000.3.8.2-12-g85770e0b
BWA mem (version 0.7.17-r1188) was used to align paired-end ChIP-exo reads against the mm10 reference genome using the following arguments: “-t 8 -v 1 -T 30 -h 5”.
Picard MarkDuplicates (version 2.7.1) was used to mark duplicate read pair alignments and then samtools view with arguments “-h -b -f 0x1 -F 0x404” was used to filter out duplicated read pairs. While read pairs were used for alignment and deduplication purposes, only read1 alignment information from the deduplicated alignments was retained for downstream analysis (read1 corresponds to the ends of the immunoprecipitated DNA fragments that are subject to exonuclease digestion, whereas read2 information has the same resolution as ChIP-seq).
Initial peak-finding was performed for each ChIP-exo experiment with ChExMix (version 0.52) with the following settings: “--threads 8 --scalewin 1000 --mememinw 6 --mememaxw 18 --q 0.05 --epsilonscale 0.5”, excluding ENCODE blacklist regions, and using MEME (version 4.11.3) internally to discover motifs associated with binding subtypes. To encourage consistency in shared peak coordinates across Fox TFs, we re-ran ChExMix in a mode where the possible binding subtype motifs are pre-initialized and remain fixed during training, using settings: “--threads 8 --scalewin 1000 --q 0.05 --epsilonscale 0.75 --motfile $motifs –noupdateinitmotifs”. The motif set provided as “$motifs” contain the MEME-discovered subtype motifs from the most populous binding subtypes in the initial ChExMix runs.
Assembly: mm10
Supplementary files format and content: bed: BED format files containing ChExMix peak call locations
Supplementary files format and content: bw: bigwig format files with read coverage of mapped ChIP-exo data
Library strategy: ChIP-exo
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| Submission date |
Sep 30, 2023 |
| Last update date |
Oct 04, 2023 |
| Contact name |
Shaun Mahony |
| E-mail(s) |
mahony@psu.edu
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| Phone |
814-865-3008
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| Organization name |
Penn State University
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| Department |
Biochemistry & Molecular Biology
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| Lab |
Shaun Mahony
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| Street address |
404 South Frear Bldg
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| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE244410 |
Joint sequence and chromatin neural networks characterize the differential abilities of Forkhead transcription factors to engage inaccessible chromatin (ChIP-exo) |
| GSE244411 |
Joint sequence and chromatin neural networks characterize the differential abilities of Forkhead transcription factors to engage inaccessible chromatin |
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| Relations |
| BioSample |
SAMN37628129 |
| SRA |
SRX21953860 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7815577_28206_FoxL2_F1804_ESC_Rosa26pr-DoxRepr-FoxL2_Dox-LIF_NoDox-Dif-24h_XO.filtered.bw |
220.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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