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| Status |
Public on Aug 20, 2024 |
| Title |
UCLA1 primed hESC, ZIC2-/- +sgZIC3, H3K27ac CUT&Tag |
| Sample type |
SRA |
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| Source name |
Primed human embryonic stem cells
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Primed human embryonic stem cells
cell line: UCLA1 primed hESCs
cell type: Human embryonic stem cells
genotype: ZIC2-/-ZIC3-/-
treatment: ZIC2-deficient primed hESCs electroporated with Cas9 and sgRNAs targeting ZIC3
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| Treatment protocol |
Cells were treated with 100 ng/ml doxycycline hyclate or DMSO of equivalent v/v concentration, where specified.
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| Growth protocol |
Primed hESCs were cultured on hESC-qualified matrigel coated plates in mTESR-plus medium. Primed hESCs were cultured in media supplemented with 10 µM Y27632 24h prior to eletroporation experiments and upon plating of electroporated cells. hESCs collected during reversion from primed to naive pluripotency were plated on a feeder layer of mouse embryonic fibroblasts and cultured in 4iLAF+0.25µM IM-12 (t5iLAF) medium for the specified period of time. Both during reversion to and upon full acquisition of naive pluripotency, hESCs were cultured in 5 % O2 and 5 % CO2. Naive hESCs were depleted of MEFs prior to collection for experiments.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed in 0.1% paraformaldehyde for 10 minutes at room temperature and the reaction was quenched with 0.14 M glycine. Cell pellets were resuspended in 200 μl cold nuclear extraction (NE) buffer (20 mM HEPES pH 7.9, 10 mM KCl, 20% glycerol, 0.1% Triton X-100, 0.5 mM spermidine, 0.15 mM spermine and protease inhibitors) and incubated for 5 minutes on ice. Nuclei were resuspended in 50 μl NE buffer and incubated with activated Concanavalin A-coated magnetic beads for 10 minutes at room temperature. Supernatant was removed using a magnetic stand and nuclei resuspended in 25 µL antibody buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.15 mM spermine, 20% glycerol and 2 mM EDTA) containing 0.5 μg primary antibody. Samples were then incubated overnight at 4 °C on a nutator. To remove unbound antibody, bead-nuclei complexes were washed twice with 150 buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.15 mM spermine, 20% glycerol) using a magnetic stand and resuspended in 25 µL 150 buffer containing 0.5 μg anti-mouse or rabbit secondary antibody for CUTANATM CUT&Tag workflows (EpiCypher). Samples were then incubated for 30 minutes at room temperature. After two washes with 150 buffer, bead/nuclei complexes were resuspended in 12 µl 1x pA/G-Tn5 pre-loaded adapter complex (EpiCypher, USA) made in 150 buffer and incubated for 1 hour at room temperature on a nutator. Bead/nuclei complexes were washed twice in 150 buffer, resuspended in 50 μl cold CUTAC-hex buffer (10 mM TAPS, 5 mM MgCl2, 10% 1,6-hexanediol, 20% glycerol and protease inhibitors) and incubated for 2 minutes at 4 °C followed by 20 minutes at 37 °C. To release pAG-Tn5 from tagmented DNA, bead/nuclei complexes were then resuspended in 5 μl 0.1% SDS and incubated for 1 hour at 58 °C. To generate libraries, 15 μl of 0.67% Triton X-100 neutralization solution was added to each sample followed by 2 μl 10 μM universal or barcoded i5 primers, 2 μl 10 μM uniquely barcoded i7 primers and 25 μl NEBNext High-Fidelity buffer (non-hot-start NEBNext formulation; NEB, USA). Samples were then placed in a thermocycler under the following cycling conditions: 5 min at 58 °C, 5 minutes at 72 °C, 45 seconds at 98 °C, 15 seconds at 98 °C (melting) and 10 seconds at 60 °C (annealing and extension). The last 2 steps were repeated for a total of 14-21 cycles followed by a 1-minute final extension at 72 °C. To purify libraries, 25 μl AMPure XP beads (Beckman Coulter, Canada) were added to the reactions and samples were incubated for 10 minutes at room temperature on a nutator. DNA was washed twice with 70% ethanol using a magnetic stand and once with 100% ethanol. DNA was eluted in 15 μl in pre-warmed 10 mM Tris pH 8 buffer using a magnetic stand.
Libraries were constructed by tagmentation such that chromatin upon treatment with Tn5 was simultaneously fragmented and tagged with Illumina-compatible sequencing adapters.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Raw reads were trimmed to 50bp using TrimGalore!
Reads were aligned against the GRCh38 reference genome using bwa mem.
Reads were filtered for unique alignments, marked for duplicates and sorted using samtools
Mitochondrial reads were removed prior to making coverage files.
Effciency biases were calculated using csaw and scale factor was calculated to make coverage tracks.
Assembly: GRCh38
Supplementary files format and content: bigWig
Library strategy: CUT&Tag
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| Submission date |
Jun 26, 2024 |
| Last update date |
Aug 20, 2024 |
| Contact name |
William A. Pastor |
| E-mail(s) |
william.pastor@mcgill.ca
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| Phone |
5143988962
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| Organization name |
McGill University
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| Department |
Biochemistry
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| Street address |
3655 Promenade Sir-William-Osler
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| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3G1Y6 |
| Country |
Canada |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE270787 |
ZIC2 and ZIC3 promote SWI/SNF recruitment to safeguard progression towards human primed pluripotency |
| GSE270822 |
ZIC2 and ZIC3 promote SWI/SNF recruitment to safeguard progression towards human primed pluripotency [CUT&Tag] |
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| Relations |
| BioSample |
SAMN42055252 |
| SRA |
SRX25061608 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8352698_U1_Z3KO_sgZ2_H3K27ac_CTag.bw |
125.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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