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| Status |
Public on Dec 31, 2020 |
| Title |
Chicken_HH3_input |
| Sample type |
SRA |
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| Source name |
Epiblast from HH3 chicken embryos
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| Organism |
Gallus gallus |
| Characteristics |
tissue: Epiblast from HH3 chicken embryos
genotype: WT
treatment: None
chip antibody: None
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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.
Cells were crosslinked in 1% of formaldehyde for 20 minutes (rotating at RT) with subsequent quenching by glycine (0.125M; rotating at RT). Washed twice with PBS and 25x Roche protease inhibitor. Then flash frozen in liquid nitrogen and stored at -80°C. Afterwards cells were thawed for ~30 minutes and lysed for 10 minutes at 4°C rotating in 50 mM Hepes, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 and 0.25% TX-100 together with protease inhibitor (Lysis Buffer 1). After centrifugation for 5 min at 2000rcf at 4°C and discarding the supernatant, pellet was resuspended in 10 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, protease inhibitor (Lysis Buffer 2) and lysed for 10 minutes at 4°C rotating. After centrifugation for 5 min at 2000rcf at 4°C and discarding the supernatant, pellet was resuspended in 10 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate and 0.5% N-lauroylsarcosine with protease inhibitor (Lysis Buffer 3). Cells were then sonicated with ActiveMotif Sonicatior (amplitude: 25%, on=20s, off=30s, 20 cycles). Debris was then centrifugated for 10 minutes, 16000rcf at 4°C. Supernatant was resuspended with 10% Triton X-100 and 10% of mixture taken for input sample. 5 ul antibody (1 ng/ul) was added to sonicated chromatin aliquot for each ChIP reaction and inverted to mix. Then rotated vertically at 4°C overnight (12-16 hrs) to bind antibody to chromatin. On the next day, 50ul (for low cell numbers) or 75ul (for higher cell numbers) of magnetic Dynabeads (Protein G) were washed three times (3X) in 1 mL cold Block Solution (0.5% BSA (w/v), 1x PBS). Antibody-bound chromatin was added to beads and inverted to mix. Then rotated vertically at 4°C for at least 4 hrs. Afterwards bound beads were washed 5X in 1 mL cold RIPA buffer (50 mM Hepes, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate). Then washed once in 1 mL TE + 50 mM NaCl on ice and centrifugated for 3 minutes at 1000rcf at 4°C to remove all remaining TE. 210 uL Elution Buffer was added to beads and DNA was eluted for 15 min at 65°C, 900 RPM, before centrifugating beads 1 min at 16000rcf at RT, adding to magnet and transferring supernatant (~200 uL) to fresh microfuge tube once beads settled. Both sample and input were then reverse crosslinked in appropriate amount of TE wash buffer, 20 mg/mL of RNAse, then Proteinase K, before extracting the DNA by phenol-chlorophorm. DNA content was measured using Qubit and the HS DNA Kit. Enrichment was measured using technical triplicates (input: 1%) by ChIP-qPCR with custom-made primers (Suppl.) on a Light Cycler 480II (Roche). All antibodies used in this study have been previously reported as suitable for ChIP: H3K4me3 (39159, Active Motif), H3K27ac (39133, Active Motif), H3K27me3 (39155, Active Motif).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
input
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| Data processing |
Basic read quality check was performed using FastQC (Babraham Bioinformatics) and read statistics were obtained with SAMtools 1.2
Reads were mapped to the mouse (mm10) and chicken (galGal6) genome reference assembly using BWA 0.7.7 and PCR dupicates were removed using Picard-Tools 2.5.0
Normalized bedGraph files were generated using DeepTools 2.5.7
Peaks were called using MACS2.1.1.20160309
Genome_build: mm10, galGal6
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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 |
GPL26853 |
| Series (2) |
| GSE160654 |
The chromatin and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo [ChIP-seq] |
| GSE160657 |
The chromatin and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo |
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| Relations |
| BioSample |
SAMN16624736 |
| SRA |
SRX9418421 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4876541_Chicken_HH3_input_nodup.bedGraph.gz |
444.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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