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| Status |
Public on Dec 31, 2020 |
| Title |
E14_H3K27me3_2 |
| Sample type |
SRA |
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| Source name |
WT E14 mESC
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| Organism |
Mus musculus |
| Characteristics |
tissue: WT E14 mESC
genotype: WT
treatment: None
chip antibody: H3K27me3 (39155, Active Motif)
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| Treatment protocol |
A full E14 WT 10cmp vial was thawed and resuspended in 10 mL of serum + LIF. The next day, cells were washed with PBS and medium was changed and the day after cells were split ¼. We began the differentiation the day after (d0). First, we trypsinized cells with 2mL TrypLE Express (Life technologies). After 5 minutes, Trypsin was quenched using 4 mL of serum + LIF. We then took 400ul for RNA extraction and the residual cells for differentiation. Both vials were centrifugated (750 and 1000 RPM respectively). Supernatant was discarded and cell pellet for RNA extraction was resuspended in Lysis Solution RL. The other vial was heavily resuspended in 5 mL of N2B27 with 0.1% of BSA (Life Technologies) and 0.1% of bFgf (Life Technologies) to get single cells. Cell resuspension and count was assessed using the BioRad TC20. We placed droplets of 15000 cells / cm2 in 10 mL of N2B27 with 0.1% of BSA and 10 ng/mL of bFgf. On d1 of differentiation, we changed medium (10 mL of N2B27 with 0.004% of BSA and 10 ng/mL bFgf) without PBS washing. On d2 of differentiation, we changed medium (10 mL of N2B27 with 0.004% of BSA, 10 ng/mL bFgf and 5 μM Xav939/Wnt inhibitor) without PBS washing. On d3 of differentiation, we changed medium (10 mL of N2B27 with 0.004% of BSA and 5 μM Xav939/Wnt inhibitor) without PBS washing. On d4 of differentiation, we changed medium (10 mL of N2B27 with 0.004% of BSA and 5 μM Xav939/Wnt inhibitor) without PBS washing. On the last day of differentiation (d5), we washed 1-2 with PBS (depending on amount of dead cells), resuspended plate in 5 mL of N2B27, scratched off cells, then extracted RNA and crosslinked these. Differentiation was assessed using RT-qPCR on a Light Cycler 480II comparing AntNPC d5 vs. E14 WT d0 relative gene expression levels (using the 2ΔCt method) with house keeping gene (Eef1a1), pluripotency markers (Pou5f1/Oct4 and Nanog), mesoderm marker (T) and ectoderm markers (Six3 and Lhx5). Standard deviations were calculated from technical triplicate reactions and were represented as error bars. Primers used can be found in the Supplemental Table.
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| Growth protocol |
10cm plates were coated in 0.1% gelatine generally overnight. Cells (E14 WT, EED−/− and RING1a−/−RING1bfl/fl mESC) were thawed and resuspended in 10ml of medium (serum + LIF). Standard serum + LIF medium contained 500 mL Knock-out DMEM (Gibco 10829-018), 95 mL of filtered ES FBS (Gibco 16141-061), 5.9 mL of antibiotics (Hyclone SV30079.01), 5.9 mL Glutamax (Gibco 35050-038), 5.9 mL MEM NEAA (Gibco 11140-035), 4.7 mL titrated LIF (Miltenyi Biotec 130-095-777), and 1.3 mL Beta-mercaptoethanol 55 mM (Gibco 21985-023). Cells were split once (1/3 or 1/4) every two days. When they reached 80-100% confluence protein was extracted using the abcam cell nuclear protein preparation protocol for western blot. RNA was was extracted with 400ul (for 6wp) or 1 mL (for 10cm plates) of Lysis Solution RL (Guanidinium Thiocyanate; analytik jena), stored at -80°C or then purified using the innuPREP DNA/RNA Mini Kit (analytik jena) according to the manufacturer’s instructions, and reverse transcribed with ProtoScript II First Strand cDNA Synthesis Kit (E6560L, NEB). For RING1a−/−RING1bfl/fl mESC cells we added 1000x Tamoxifen (OHT) for 72h right after first passage. Floxed deletion was assessed after ChIP / HiChIP lysis in electrophoresis gel using flanking primers. Deleted protein was assessed via standard Western blot (using plate reader).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fertilized chicken eggs (white leghorn; Gallus gallus domesticus) were obtained from a local breeder (LSL Rhein-Main) and incubated at 37°C and 80% humidity in a normal poultry egg incubator (Typenreihe Thermo-de-Lux). Following microsurgical procedures, the eggs were re-incubated until the embryos reached the desired developmental stages. The developmental progress was determined according to the staging system of HH (Hamburger and Hamilton, 1992). Mice were housed and bred under standard conditions in the CECAD ivRF. The breedings described were approved by the Landesamt für Natur, Umwelt, und Verbraucherschutz Nordrhein-Westfalen (LANUV), Germany (animal application 84-02.04.2015.A405). Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibodies againts the specific proteins of interest in each case.
HiChIP was performed as described by Mumbach et al. 2016 Nat Methods with some modifications. We generally replaced the ChIP protocol with the one described above, we cut and ligated overnight (NEB T4 Ligase, instead of Invitrogen T4) and DNA was extracted with phenol-chlorophorm not a kit. For low cell numbers, we increased the centrifugation time to 30 minutes and 15 minutes after lysis, as well as 30 minutes after ligation to see a pellet more accurately. Generally 12 cycles were used for Tn5 Nextera PCR amplification (Illumina Nextera DNA UD Indexes Kit). We aimed for 100M read pairs for each run on a NovaSeq 6000 sequencer (Illumina).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
biological replicate 2
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| Data processing |
Library strategy: HiChIP
Basic read quality check was performed using FastQC (Babraham Bioinformatics) and read statistics were obtained with SAMtools 1.2
Reads were mapped to the human (hg19), mouse (mm10) or chicken (galgal6) genome reference assembly using Bowtie2 within the HiC-Pro 2.10 framework.
Normalized bedGraph files were generated using hichipper 0.7.3 and DeepTools 2.5.7
Peaks were called using MACS2.1.1.20160309 and used as input for loop calling by FitHiChIP 7.0
Valid pairs were converted using cooler 0.8.7 and the custom scripts within HiC-Pro 2.10, then loops were visualized with coolpup.py 0.9.2, hicexplorer 3.0 and HiCPlotter 0.8.1.
Genome_build: hg19, galGa6, mm10
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| Submission date |
Nov 02, 2020 |
| Last update date |
Dec 31, 2020 |
| Contact name |
Giuliano Crispatzu |
| Organization name |
University of Cologne (UoC), Germany
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| Department |
CECAD
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| Street address |
Joseph-Stelzmann-Straße 26
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| City |
Cologne |
| ZIP/Postal code |
50931 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE160656 |
The chromatin and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo [HiChIP] |
| GSE160657 |
The chromatin and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo |
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| Relations |
| BioSample |
SAMN16624749 |
| SRA |
SRX9418426 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4876557_E14_H3K27me3_2_FitHiChIP_L_COV.interactions_FitHiC.bed.gz |
708.3 Mb |
(ftp)(http) |
BED |
| GSM4876557_E14_H3K27me3_2_allValidPairs.txt.gz |
1.2 Gb |
(ftp)(http) |
TXT |
| GSM4876557_E14_H3K27me3_2_temporary_control_lambda.ALL.clipped.bedGraph.gz |
499.9 Mb |
(ftp)(http) |
BEDGRAPH |
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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