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| Status |
Public on Mar 23, 2022 |
| Title |
Naïve_UNC1999_H3K27me3_Replicate_1 |
| Sample type |
SRA |
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| Source name |
Human embryonic stem cells (ESCs) and Drosophila Schneider 2 cells (S2)
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| Organisms |
Drosophila melanogaster; Homo sapiens |
| Characteristics |
cell type: Naive ESCs (H9 line) and Drosophila S2
cut and run antibodies: Tri-Methyl-Histone H3 (Lys27) (Cell Signaling Technology, 9733)
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| Treatment protocol |
2.5 μM UNC1999 was applied to primed hPSCs the day after passaging for 4 days. 1 μM UNC1999 was applied to naïve hPSCs the day after passaging for 4 days.
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| Growth protocol |
Human pluripotent stem cells (hPSCs) were grown in a 5% O2 and 5% CO2 incubator under humidified conditions at 37 °C. H9 primed hPSCs were cultured in feeder-free conditions on plates pre-coated with Vitronectin in complete TeSR-E8™ medium. H9 chemically reset naive hPSCs were cultured on Geltrex-coated plates in PXGL medium, which consists of a 1:1 mixture of DMEM/F12 and Neurobasal, 0.5x N2-supplement, 0.5x B27-supplement, 2 mM L-Glutamine, 1x penicillin-streptomycin, 0.1 mM β-mercaptoethanol, 1 μM PD0325901, 2 μM XAV939, 2 μM Gö6983 and 20 ng/mL human LIF. Drosophila S2 cells were grown in a non-humidified incubator at 28°C without additional CO2 in normoxic conditions. Drosophila S2 were cultured in T75 flasks in Schneider’s Drosophila medium supplemented with 10% heat inactivated FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Concanavalin A conjugated paramagnetic beads were resuspended and washed twice on a magnetic rack in a bead activation buffer composed of: 20 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl2 and 1 mM MnCl2. Naive or primed hPSCs and Drosophila S2 cells were dissociated and counted. Cell pellets were then washed twice with a wash buffer composed of: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine and supplemented with EDTA-free protease inhibitor. 50,000 human cells and 20,000 Drosophila cells were added in wash buffer to Concanavalin A beads and incubated for 10 min at RT with rotation to immobilise cells. Concanavalin A beads were then collected with a magnetic rack, the supernatant discarded and beads were resuspended in antibody buffer, composed of wash buffer supplemented with 0.08% Digitonin and 2 mM EDTA. 1 μg of antibody was added to beads which were then incubated overnight at 4 °C with rotation to allow permeabilisation and target binding. Antibodies used were: rabbit anti-Tri-Methyl-Histone H3 (Lys27) (Cell Signaling Technology, 9733), rabbit anti-IgG (Invitrogen, 31188) and anti-Histone H2Av spike-in antibody (Active Motif, 61686). The following day, beads were washed twice with digitonin buffer, composed of wash buffer supplemented with 0.08% Digitonin. To 50 μl digitonin buffer, 2.5 μl CUTANA™ pA/G MNase (EpiCypher, 15-1016) was added and incubated for 10 min at RT to promote pA/G MNase binding to the antibody constant region. Beads were again washed twice with digitonin buffer and MNase was activated with 2 mM CaCl2 and incubated for 2 h at close to 0 °C to facilitate chromatin fragmentation under sites of antibody binding. MNase activity was terminated by adding 100 μl stop buffer, composed of 340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/ml RNase A and 50 μg/ml Glycogen. Cleaved DNA fragments were released from nuclei by incubation for 10 min at 37 °C, spinning for 5 min at 16,000 xg at 4 °C and collecting the supernatant from the beads on a magnetic rack. DNA was then purified by incubation with 1 μl SDS (20 %) and 1.5 μl Proteinase K (20 mg/ml) at 70 °C for 10 min, followed by a 1.8x AMPure XP bead (Beckman Coulter) clean-up into DNA lo-bind tubes (Eppendorf) and eluted in 50 μl 0.1x TE.
Libraries were prepared using the NEBNext Ultra II DNA library preparation kit for Illumina (NEB) using the manufacturer’s protocol, with libraries indexed using NEBNext Multiplex Oligos for Illumina (Index Primers Set 1 and Set 2) (NEB). Following library preparation, library fragment size and concentration were determined using the Qubit fluorometer double stranded DNA high sensitivity assay kit, using an Agilent Bioanalyzer 2100 and by KAPA Library Quantification Kit (KAPA Biosystems). Samples were sequenced on an Illumina NextSeq500 instrument as HighOutput 75 bp paired-end reads at the Babraham Institute Next Generation Sequencing Facility.
CUT&RUN
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Standard Illumina base-calling pipeline.
Reads were trimmed using trim galore v0.6.5 using default parameters to remove the standard Illumina adapter sequence, and mapped to the human genome GRCh38 and Drosophila genome BDGP6 using Bowtie2. BAM files were imported into SeqMonk.
Genome_build: GRCh38 and BDGP6
Supplementary_files_format_and_content: A tab-separated file of log2 RPM read counts per 1kb. Fields are chomosome, start, end, log2 RPM values for each sample.
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| Submission date |
Sep 08, 2021 |
| Last update date |
Mar 23, 2022 |
| Contact name |
Vincent Pasque |
| E-mail(s) |
vincent.pasque@kuleuven.be
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| Organization name |
KU Leuven
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| Department |
Development and Regeneration
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| Street address |
Herestraat 49 bus 804
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| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL22339 |
| Series (2) |
| GSE176175 |
Integrated Multi-Omics Reveal Polycomb Repressive Complex 2 Restricts Human Trophoblast Induction. |
| GSE183735 |
CUT & RUN H3K27me3 of Human Naïve and Primed Pluripotent Stem Cells with and without EZH2 inhibition. |
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| Relations |
| BioSample |
SAMN21363160 |
| SRA |
SRX12118218 |
| 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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