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| Status |
Public on Jul 15, 2018 |
| Title |
RRBS from Mbd3f/- system day8 |
| Sample type |
SRA |
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| Source name |
Mbd3f/- cell line, Day8 post DOX induction
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| Organism |
Mus musculus |
| Characteristics |
tissue: Mbd3f/- cell line
stage of reprogramming: Day8
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 |
Genomic DNA was extracted from snap-frozen cell pellets (Quick-gDNA MiniPrep, Zymo Research).
300ng of DNA was digested overnight with MspI (NEB R0106) followed by end-repair (NEB E6050) and addition of a 3’ overhang A using Klenow fragment (3'-5' Exo-, NEB M0212). The resulting DNA fragments were size-selected for ones shorter than 500bp using AMPure XP beads (Beckman Coulter A63881). 10ng of size-selected DNA was ligated, using quick T4 ligase (NEB M2200), into a cut and T-tailed plasmid containing a 6bp unique molecular identifier (UMI) sequence. The ligated plasmid was bisulfite converted and cleaned (EZ DNA methylation Gold kit, Zymo Research). The converted DNA was amplified using 25 cycles of PCR with GoTaq Hot Start polymerase (Promega M5005) and primers matching specific locations on the plasmid. The primer sites were cut from the amplicons using XbaI (NEB R0145) end EcoRI-HF (NEB R3101) followed by end-repair (NEB E6050). Further details regarding the protocol and the plasmid are available in Shipony Z et al. Dynamic and static maintenance of epigenetic memory in pluripotent and somatic cells. Nature 2014 Sep 4;513(7516):115-9. RRBS fragments underwent addition of a 3’ overhang A using Klenow fragment (3'-5' Exo-, NEB M0212). The fragments were then ligated to Illumina compatible adapters using quick T4 ligase (NEB M2200) and amplified using 8 cycles of PCR with GoTaq Hot Start polymerase (Promega M5005) and primers matching the Illumina adapters. The final products underwent gel electrophoresis using NuSieve 3:1 agarose (Lonza 50094). Gel bands matching fragment sizes of 200-500bp were cut and the DNA was extracted (MinElute Gel Extraction Kit, Qiagen 28604).
To improve cluster registration at the Illumina sequencer, paired-end sequencing was performed as following: 1. Sequencing of read 1 was performed with 22 dark cycles and 80 regular cycles (resulting in a read of bases 23-102 of read 1). The bases are included in the R1 files. 2. Sequencing of read 1 was performed with regular dark cycles (resulting in a read of bases 1-22 of read 1). The bases are included in the R2 files. 3. Sequencing of read 2 was performed normally with 80 regular cycles (bases 1-80 of read 2). The bases are included in the R3 files.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
FASTQ files were generated using the Illumina CASAVA 1.8.2 software.
The plasmid introduces a CTAGANNNNNNATTGATT prefix at one read and an AATTCAATT prefix on the other read (were NNNNNN is the UMI). These prefixes were removed and the UMI recorded for each fragment. A small fraction of reads include longer prefixes that result from amplicons that did not undergo digestion by XbaI or EcoRI. These reads were identified and the longer prefixes were removed as well.
The reads were aligned to the mm9 reference genome using bismark version 0.7.7 with bowtie 1.0.1. Alignment was performed independently for each read of the read-pair. We expect each read to start at an MspI restriction site. Therefore we discarded reads that failed to align within 1bp of such sites. We also discarded read-pairs where each read aligned to remote MspI sites. On the other hand, in cases where one read uniquely mapped on a restriction site but its pair could not be mapped uniquely or could not be mapped at all, we attempted to re-align the entire read pair to the fragment.
To avoid PCR biases we grouped reads that map to the same MspI restriction site, have the same UMI and were ligated into the plasmid in the same orientation (positive or negative). Only the first such read was maintained and all other reads were dropped.
The methylation state of each CpG was extracted from the bismark output. For each CpG, the number of reads in which it was methylated or unmethylated was counted and recorded.
Genome_build: mm9
Supplementary_files_format_and_content: Tab-separated text files containing CpG methylation values. The files' columns are: Column1: chromosome. Column 2: position of CpG (1-based). Column 3: seen count. Column 4: methylation count. Note that each CpG's coverage is given by column 3, and its methylation value by column4/column3.
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| Submission date |
Dec 08, 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 |
GPL18480 |
| Series (2) |
| GSE102518 |
High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems |
| GSE107853 |
High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems [RRBS] |
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| Relations |
| BioSample |
SAMN08150391 |
| SRA |
SRX3459390 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2881847_RRBS_Mbd3f-_day8.cpgs.txt.gz |
7.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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