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| Status |
Public on Feb 01, 2022 |
| Title |
ChIPseq NELF in Zbtb11KO Zfp131KO ES cells (iZbtb11-HA iZfp131-V5 Zbtb11KO Zfp131KO) replicate 1 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: Ainv15(iZbtb11-HA iZfp131-V5 Zbtb11KO Zfp131KO)
chip target: NELF
chip antibody: anti-NELF (Sigma HPA007187)
treatment: 3d without doxycycline in -2i+LIF
cell type: Embryonic stem cells
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| Treatment protocol |
Wildtype, iZbtb10-HA Zbtb10KO, iZbtb11-HA Zbtb11KO, iZfp131-HA Zfp131KO cells, and iZbtb11-HA iZfp131-V5 Zbtb11KO Zfp131KO cells were grown in the absence of doxycycline in +2i+LIF or -2i +LIF media for 3 days.
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| Growth protocol |
ESCs were cultured on 0.1% gelatin (Milipore) coated dishes at 37 °C, 8% CO2 in ESC medium (Advanced DMEM/F12: Neurobasal (1:1) Medium (Gibco), supplemented with 2.5% mESC-grade fetal bovine serum (Corning), 1x N2 (Gibco), 1x B27 (Gibco), 2 mM L-glutamine (Gibco), 0.1 mM ß-mercaptoethanol (Gibco)) supplemented with LIF (leukemia inhibitory factor, 1,000 U ml–1 (Millipore)) and 2-inhibitor cocktail (3 mM CHIR (BioVision) and 1 mM PD0325901 (Sigma)) unless stated otherwise.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Wildtype and mutant cells were collected after being grown for 3d in +2i or -2i conditions in the presence or absence of 3 ng/uL doxycycline. 1 mM DSG (ProteoChem) was used for crosslinking at RT for 15min. 1% Formaldehyde (vol/vol) was added for an additional 15min until quenched with glycine (Sigma). Cells were separated into aliquots of 25x106 and centrifuged to freeze pellets at -80°C. Thawed cells were lysed in 5mL of 50 mM HEPES-KOH pH 7.5 (Gibco), 140 mM NaCl (Thermo Fisher), 1 mM EDTA pH 8.0, 10% glycerol (vol/vol) (Thermo Fisher), 0.5% Igepal (vol/vol) (Sigma), 0.25% Triton X-100 (vol/vol) with 1×protease inhibitors (Roche) for 10min at 4°C. Cells were centrifuged for 5min at 1100 rpm and resuspended in 5mL of 10 mM Tris-HCl pH 8.0 (Boston BioProducts), 200 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0 (Boston BioProducts) with 1×protease inhibitors, and incubated for an additional 10 min at 4°C on a rotating platform. Cells were centrifuged for 5min at 1100 rpm and resuspended in 2mL of Sonication Buffer (50 mM Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100 (vol/vol), 0.1% sodium deoxycholate (wt/vol), 0.1% SDS (vol/vol) with 1×protease inhibitors). Sonication was performed in two Bioruptor tubes per sample with 0.45 g sonication beads. Bioruptor Pico (Diagenode) was used for 18 cycles of 30 sec on and 30 sec off to sonicate to an average size of approximately 200 bp. To immunoprecipitate, Dynabeads protein-G (Thermo Fisher) and antibodies (HA, H3K4me3, H3K27me3, H3K27ac, SIN3A, CHD4) were incubated with sonicated chromatin for 16h at 4°C on a rotating platform. After the immunoprecipitation, the sample was washed with sonication buffer, high salt sonication buffer (500 mM NaCl, LiCl wash buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl (Sigma), 0.5% Igepal (vol/vol), 0.5% sodium deoxycholate (wt/vol)), and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8). The sample was eluted in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS (vol/vol)) by incubating for 45min at 65°C. Crosslinks were reversed by incubating the sample for 16h at 65°C. RNA was removed by 0.2 mg/mL RNAse A (Sigma) in 200 μL of TE for 2h at 37°C. To remove protein, 0.2 mg/mL Proteinase K (Invitrogen) and CaCl2 (Sigma) were added and the sample incubated at 55°C for 30min. DNA was first purified with phenol:chloroform:isoamyl alcohol (25:24:1; vol/vol) (Invitrogen) and then by performing salt-ethanol precipitation. DNA pellets were resuspended in 50 uL H2O.
Illumina DNA sequencing libraries were prepared with half of the ChIP sample and a 1:100 dilution of the input sample in H2O. Library preparation was performed with end repair, A-tailing and ligating multiplexed adapters (Illumina-compatible Bioo Scientific). Agencourt AmpureXP beads (Beckman Coulter) were used to remove unligated adapters. Libraries were then amplified by PCR with TruSeq primers (Sigma) and Phusion polymerase (New England Biolabs). Libraries with sizes ranging between 250-550bp were gel purified (Qiagen). KAPA library amplification kit was used on Roche Lightcycler 480 to quantify the library prior to sequencing. Libraries were sequenced on Illumina NextSeq 500 using V2.5 chemistry (75 cycles, single-end 75bp) at the Genomics Core Facility at NYU.
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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 |
Fastq files were aligned to the mouse genome (mm10) using Bowtie (v1.0.1) with options “-q–best–strata -m 1–chunkmbs 1024”.
MultiGPS (v.0.74) was used to call peaks after alignment on all transcription factors. A q-value cutoff of <0.01 was used to identify significant binding events. Histone modification and ATAC-seq peaks were called using the DomainFinder module in SeqCode (https://github.com/seqcode/seqcode-core/blob/master/src/org/seqcode/projects/seed/DomainFinder.java). Contiguous 50-bp genomic bins with significantly higher read enrichment compared with normalized input were identified (binomial test, p <0.05).
Differential binding and modification analyses were done using DESeq2 (v1.28.1) with an adjusted p-value cutoff of <0.05 and all default options. To create the count matrix for DESeq2, peaks were first called independently in the wildtype and knockout ChIP-seq datasets. Overlapping peak regions were merged between the wt and KO. Peak regions found exclusively in either file were also included. The final count matrix was created using Bedtools coverage with the “-counts” option separating each replicate into its own count column. Before running DESeq2 on the matrix, counts were normalized by the length of the corresponding region for histone modifications (domains have differing lengths).
Genome_build: mm10
Supplementary_files_format_and_content: MultiGPS: MultiGPS output files (text format)
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| Submission date |
Jan 31, 2022 |
| Last update date |
Feb 01, 2022 |
| 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 |
GPL24247 |
| Series (2) |
| GSE160959 |
The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by pausing POL II at pro-differentiation genes [ChIP-Seq] |
| GSE160966 |
The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by pausing POL II at pro-differentiation genes |
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| Relations |
| BioSample |
SAMN25519994 |
| SRA |
SRX13999589 |
| 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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