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| Status |
Public on Oct 31, 2019 |
| Title |
6h_No_Triptolide_1 |
| Sample type |
SRA |
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| Source name |
6h release into S-phase, no treatment with Triptolide
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| Organism |
Mus musculus |
| Characteristics |
tissue: Embryonic Stem Cell
genotype: KH2: 13 Flag-BAP-H3 tags
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| Treatment protocol |
For G1 synchronization experiments, mESCs were plated at 50-60% confluency and pulsed for 18 hr with 8 µM Thymidine, followed by a PBS wash and a 7-8 hr release into 2i Tet-free media. mESCs were then given a second Thymidine treatment at a 5-6 µM concentration for 12-13 hr. For the biotinylation experiments, a 6 hr pulse of 2 µg/ml of doxycycline and 1µg/ml of exogenous biotin was given during the latter half of the second Thymidine treatment and cells were harvested at the G1/S-block (time 0h) or through one DNA replication (time 12h). For Retinoic Acid (RA) samples, 1uM of RA was added before double Thymidine synchronization.
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| Growth protocol |
KH2 mESCs were grown in DMEM supplemented with 15% FBS (for CRISPR/Cas9 targeting) or Tet-free FBS (Gemini 100-800, for biotinylation experiments), L-glutamine, penicillin/streptomycin, non-essential amino acids, 0.1 mM β-mercaptoethanol, LIF, and 2i inhibitors which includes 1 µM MEK1/2 inhibitor (PD0325901) and 3 µM GSK3 inhibitor (CHIR99021).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For native MNase chromatin preparation, nuclei were harvested from mESCs by hypotonic lysis TMSD buffer (40 mM Tris, 5 mM MgCl2, 0.25 M Sucrose with protease inhibitors), pelleted for 10 min at 3600 rpm at 4° C. Nuclei were solubilized by resuspending in NIB-250 (15 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM Sucrose with protease inhibitors) containing 0.3% NP-40. A chromosome pellet was isolated by centrifuging the solubilized nuclei for 5 min at 600 rcf at 4° C. The chromosome pellet was washed with NIB-250 buffer, then resuspended with MNase digestion buffer (10 mM Hepes, 50 mM NaCl, 5 mM MgCl2, 5 mM CaCl2 with protease inhibitors) and treated with MNase (Sigma) until a DNA fragment size of 150-500 bp (1-3 nucleosomes) was attained. MNase digestion was stopped with 15 µl of EGTA (0.5 M) per 1 ml pellet and then spun at 600 rcf for 5 min at 4° C. The supernatant was placed in a fresh tube and the chromatin pellet was further processed by adding the same volume of BC500 (40 mM Tris, 5 mM MgCl2, 500 mM NaCl and 5% glycerol, and protease inhibitors) and EGTA (0.5 M) as in the MNase buffer plus EGTA step. The soluble chromosomes in BC500 and EGTA were then incubated for 30 min while rotating at 4° C, allowing for further enrichment of desired chromatin. Samples were pelleted at 600 rcf and the BC500 supernatant was pooled with an equal volume of MNase-treated supernatant to acquire the starting chromatin material for chromatin immunoprecipitations (IPs).
For ChIP-seq, libraries were prepared by standard protocol. First, fragments are repaired to generate blunt 5' P ends using 'EndIT DNA End Repair kit' (Epicentre). Second, 3' A overhang was generated using 'Klenow Fragment (3´→ 5´ exo-)' (NEB). Third, Illumina barcodes were ligated using 'Rapid T4 DNA Ligase' (Enzymatics). Lastly, libraries were size selected using 'Ampure beads' (Beckman Coulter), then amplified, quantified and pooled. For RNA-seq, polyA-beads were used to purify mRNA followed by cDNA synthesis prior to standard library constructions as described above.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
6h release into S-phase, no treatment with Triptolide
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| Data processing |
Quality of sequencing was assessed with FastQC v0.11.4 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads having less than 80% of quality scores above 25 were removed with NGSQCToolkit v2.3.3
Reads were aligned to the mouse mm10 reference genome from Illumina igenomes UCSC collection using bowtie v1.0.0 allowing 3 mismatches and keeping uniquely aligned reads
Sam ouputs were converted to Bam with Samtools v1.0.6
Data were further processed with Pasha v0.99.21 with the following parameters: WIGvs = TRUE, incrArtefactThrEvery = 7000000, elongationSize = NA.
Fixed steps wiggle files were converted to bigwigs with the script wigToBigWig available on the UCSC Genome Browser website (http://hgdownload.soe.ucsc.edu/admin/exe/)
The wiggle files of 50 bp bins were converted to 200 bp bins with an in-house script
Spike-in normalization was achieved using Drosophila Melanogaster DNA aligned to Illumina igenomes dm3
Genome_build: mm10 and dm3
Supplementary_files_format_and_content: Fastq files for raw data, bigwigs of the processed and spike-in normalized experiments are provided.
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| Submission date |
Aug 23, 2019 |
| Last update date |
Nov 01, 2019 |
| Contact name |
Danny Reinberg |
| E-mail(s) |
danny.reinberg@nyulangone.org
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| Organization name |
NYU school of Medicine
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| Department |
Biochemistry
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| Lab |
Reinberg
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| Street address |
522 first avenue
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| City |
new york |
| State/province |
new york |
| ZIP/Postal code |
10016 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE136245 |
Active and repressed chromatin domains exhibit distinct nucleosome segregation during DNA replication |
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| Relations |
| BioSample |
SAMN12627594 |
| SRA |
SRX6755623 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4043228_Salmon_6h_1.tabular.txt.gz |
2.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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