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| Status |
Public on Jan 02, 2023 |
| Title |
ChIPseq_ESC_WT_EED_Rep2 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic stem cells
antibody: EED
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| Treatment protocol |
No treatment
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| Growth protocol |
TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
10 million cells were crosslinked in a 1% formaldehyde solution for 5 minutes at room temperature, after which glycine was added to a final concentration of 125 mM and incubated for 5 minutes to quench the reaction. These fixed cells were centrifuged at 2,500 g for 5 minutes at 4 °C and the pellet was washed twice with 1 mL PBS. Nuclei were extracted by incubating the fixed cells with 500 µL of cell lysis buffer (20 mM Tris-HCl pH 8.0, 85 mM KCl, 0.5% NP40) for 10 min on ice then spun down for 3 min at 2,500 g. The pellet was resuspended in nuclei lysis buffer (10 mM Tris pH7.5, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS) then sonicated on a Covaris E220 Evolution sonicator (peak incident power 140.0, duty factor 5.0, cycles per burst 200, 20 min). After sonication, chromatin was spun down at 21,000 g for 10 min to pellet insoluble material. The supernatant was transferred to a fresh tube, the volume was increased to 1 ml with chip dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl), and 5 µg of EED antibody was added (Abcam ab240650). Immunoprecipitation was carried out at 4 ºC overnight with gentle rotation followed by incubation with protein A dynabeads (Thermo Scientific) for 4 hours the next day. This was followed by two washes of each of the following: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl); high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris, pH 8.1, 500 mM NaCl); LiCl wash buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and TE buffer pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA pH 8.0). DNA was eluted twice using 50 µl of elution buffer (0.5–1% SDS and 0.1 M NaHCO3) at 65 °C for 15 min. A 16-µl volume of reverse crosslinking salt mixture (250 mM Tris-HCl, pH 6.5, 62.5 mM EDTA pH 8.0, 1.25 M NaCl, 5 mg ml−1 Proteinase K) was added, and samples were allowed to incubate at 65 °C overnight. The next day 284 µl TE was added to the reaction along with 400 µl phenol-chloroform-isoamyl alcohol (Invitrogen), the solution was briefly vortexed, and then centrifuged at 21,000 g for 10 minutes at room temperature in phase lock tubes (VWR). After centrifugation, the aqueous phase was collected and combined with 20 µL 5M NaCl, 1 µL 20 mg/mL glycogen (Thermo Scientific), and 880 µL 100% ethanol. This solution was stored at -20 ºC overnight and then centrifuged at 21,000 g 4 ºC for 1 hour the next day. This was followed by two washes with 70% ethanol and elution of the pelleted DNA in 50 µL 1x TE.
Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) following the manufacturer’s protocol. All libraries were sequenced using 100 bp paired-end sequencing (200 cycles kit) on a NovaSeq 6000 platform.
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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 |
Raw reads of ESC and TSC EED ChIP-seq samples as well as their respective input samples were subjected to adapter and quality trimming with cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25 --adapter AGATCGGAAGAGC -A AGATCGGAAGAGC).
Reads were aligned to the mouse genome (mm10) using BWA with the ‘mem’ command (version 0.7.17, default parameters).
A sorted BAM file was obtained and indexed using samtools with the ‘sort’ and ‘index’ commands (version 1.10).
Duplicate reads were identified and removed using GATK (version 4.1.4.1) ‘MarkDuplicates’ and default parameters.
Replicates of treatment and input samples were merged respectively using samtools ‘merge’.
Peaks were called using MACS2 ‘callpeak’ (version 2.1.2; parameters --bdg --SPMR --broad) based on merged replicates using the input samples as control samples and only peaks with a q-value < 0.01 were considered for downstream analyses.
Genome-wide coverage tracks for single and merged replicates normalized by library size were computed using deepTools bamCoverage (parameters: --normalizeUsing RPGC --extendReads).
Coverage tracks were subtracted by the respective input using deeptools ‘bigwigCompare’.
Assembly: mm10
Supplementary files format and content: Bigwig coverage files (RPGC normalization, input subtracted).
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| Submission date |
Nov 02, 2022 |
| Last update date |
Jan 02, 2023 |
| Contact name |
Sara Hetzel |
| E-mail(s) |
hetzel@molgen.mpg.de
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| Organization name |
Max Planck Institute for Molecular Genetics
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| Department |
Genome Regulation
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| Lab |
Meissner Lab
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| Street address |
Ihnestraße 63
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| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE166362 |
Dynamic antagonism between key repressive pathways maintains the placental epigenome |
| GSE217134 |
Dynamic antagonism between key repressive pathways maintains the placental epigenome (ChIP-seq) |
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| Relations |
| BioSample |
SAMN31576996 |
| SRA |
SRX18127737 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6705545_ChIPseq_ESC_WT_EED_Rep2_RPGC_mm10_input_subtracted.bw |
247.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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