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| Status |
Public on Jul 15, 2018 |
| Title |
ATAC-seq from Mbd3f/- system, IPS |
| Sample type |
SRA |
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| Source name |
Mbd3f/- cell line, Established induced pluripotent stem cells
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| Organism |
Mus musculus |
| Characteristics |
tissue: Mbd3f/- cell line
stage of reprogramming: IPS
genotype: Mbd3f/-
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| Treatment protocol |
Reprogramming dynamics samples were generated by adding Dox to Secondary MEF. ES and iPSC samples were harvested after 3-4 days of growth in N2B27 2i\LIF medium. Reprogramming samples of days 3-8, ES and iPSC samples were plated on irradiated human foreskin fibroblasts (HFF).
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| Growth protocol |
Maintenance and reprogramming (Day 3 and beyond) of murine naïve pluripotent cells were conducted in bovine serum-free N2B27-based media or KSR media: N2B27- based media: 500ml KO-DMEM (Invitrogen), 5ml N2 supplement (Invitrogen; 17502048), 5ml B27 supplement (Invitrogen; 17504044), 15% knockout serum replacement (Invitrogen – 10828), 1mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Sigma), penicillin-streptomycin (Invitrogen), 5mg/ml BSA (Sigma). KSR media: 500 ml DMEM (Invitrogen) , 1mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Sigma), penicillin- streptomycin (Invitrogen). Naïve conditions for murine PSCs included 10μg recombinant human LIF (Millipore; LIF1005). Where indicated 2i was added 48 hours after OSKM induction: small-molecule inhibitors CHIR99021 (CH, 3μM- Axon Medchem) and PD0325901 (PD 0.2 or 1μM - Axon Medchem).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
50,000 cells were centrifuged (500g, 3 minutes), then washed using 50 μL of cold PBS, and centrifuged again (500g for 3 min). Cells were lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500g for 10 min using a refrigerated centrifuge. Next, the pellet was re-suspended in the transposase reaction mix (25 μL 2× TD buffer, 2.5 μL transposase (Illumina) and 22.5 μL nuclease-free water). The transposition reaction was carried out for 30 minutes at 37 °C and immediately transfered to ice. The sample was then purified using Qiagen MinElute kit.
Following purification, the library fragments were amplified using custom Nextera PCR primers 1 and 2 for a total of 12 cycles. Following PCR amplification the libraries were purified using a Qiagen MinElute Kit.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
We used Illumina CASAVA 1.8.2 software to generate fastq files.
Reads were aligned to mm10 mouse genome using Bowtie2 with the parameter -X2000 (allowing fragments up to 2 kb to align).
Duplicated aligned reads were removed using Picard MarkDuplicates tool with the command REMOVE_DUPLICATES=true.
To identify chromatin accessibility signal we considered only short reads (≤ 100bp) that correspond to nucleosome free region.
To detect and separate accessible loci in each sample, we used MACS version 1.4.2-1 with --call-subpeaks flag (PeakSplitter version 1.0).
93,137 enhancers with co-localized ATAC-seq and H3K27ac peaks were identified.
For each promoter and each enhancer we report here the read depth in a 50-bp bins, 1 Kb around the TSS or 300 bp around the enhancer summit.
Genome_build: mm10
Supplementary_files_format_and_content: Promoter "matrix" files contain for each TSS, the number of reads in 50bp bins, spanning 1Kb window around the TSS. 1st column - gene esemble ID, columns 2-21 - read num in 50bp bins. Enhancer "matrix" files contain for each identified enhancer, the number of reads in 50bp bins, spanning 300bp window around the enhancer summit. 1st column - enhaner genomic interval. 2nd column - enhancer ID number, columns 3-8 - read num in 50bp bins.
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| Submission date |
Sep 13, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Noa Novershtern |
| E-mail(s) |
noa.novershtern@weizmann.ac.il
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| Organization name |
Weizmann Institute of Science
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| Department |
Molecular Genetics
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| Street address |
Weizmann Institute
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| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE102518 |
High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems |
| GSE103821 |
High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems [ATAC-Seq] |
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| Relations |
| BioSample |
SAMN07638592 |
| SRA |
SRX3182574 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2782411_ATAC_iPS.enhancer_150.matrix.txt.gz |
1.8 Mb |
(ftp)(http) |
TXT |
| GSM2782411_ATAC_iPS.prom_500.matrix.txt.gz |
2.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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