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Sample GSM2739969 Query DataSets for GSM2739969
Status Public on Jul 15, 2018
Title H3K4me3 ChIP-seq, in Mbd3f/- system, Day2
Sample type SRA
 
Source name Mbd3f/- cell line, Day2 post DOX induction
Organism Mus musculus
Characteristics cell line: Mbd3f/- cell line
target protein: H3K4me3
antibody: Abcam 8580
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).
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).
Extracted molecule genomic DNA
Extraction protocol Approximately 25*10^6 cells were cross-linked in formaldehyde (1% final concentration, 10 min at room temperature (RT)), and then quenched with glycine (5 min at RT). Fixed cells were lysed in 50mM HEPES KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40 alternative, 0.25% Triton supplemented with protease inhibitor at 4°C (Roche, 04693159001), centrifuged at 950 x g for 10 min and re-suspended in 0.2% SDS, 10mM EDTA, 140mM NaCl and 10mM Tris-HCL. Cells were then fragmented with a Branson Sonifier (model S-450D) at -4°C to a size range between 200 and 800 bp, and precipitated by centrifugation. 10 ug of antibody were pre-bound by incubating with Protein-G Dynabeads (Invitrogen100-07D) in blocking buffer (PBS supplemented with 0.5% TWEEN and 0.5% BSA) for 2 hours at room temperature. Washed beads were added to the chromatin lysate, and then incubated overnight. Samples were washed 5 times with RIPA buffer, twice with RIPA buffer supplemented with 500 mM NaCl, twice with LiCl buffer (10 mM TE, 250mM LiCl, 0.5% NP-40, 0.5% DOC), once with TE (10Mm Tris-HCl pH 8.0, 1mM EDTA), and then eluted in 0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris Hcl pH 8.0 at 65°C. Eluate was incubated in 65°C for 4 hours, and then treated sequentially with RNaseA (Roche, 11119915001) for 30 min and Proteinase K (NEB, P8102S) for two hours. DNA was purified with The Agencourt AMPure XP system (Beckman Coulter Genomics, A63881).
120ul SPRI AMPure XP beads (Agencourt) were added to the reverse-crosslinked samples, pipette-mixed 15 times and incubated for 2 minutes. Supernatant were separated from the beads using a magnet for 4 minutes. Beads were washed on the magnet with 70% ethanol and then air dried for 4 minutes. The DNA was eluted in 40 ul EB buffer (10 mM Tris-HCl pH 8.0) by pipette mixing 25 times. For the remainder of the library construction process (DNA end-repair, A-base addition, adaptor ligation and enrichment) a general SPRI cleanup involves addition of buffer containing 20% PEG and 2.5 M NaCl to the DNA reaction products (without moving them from their original well position). After thorough mixing and a 2-minute incubation at room temperature, plates are transferred to a magnet plate, incubated for 4 minutes and supernatant removed. Beads are then washed on the magnet with 150ul 70% ethanol and then air dried for 4 minutes. The DNA is eluted with 40ul of EB buffer by pipette mixing 25 times. Reagent kits are prepared in advance for all enzymatic steps (New England Biolabs). The DNA end-repair was performed by adding 27 µl of a master mix (17 µl master mix (5 ul T4 buffer, 5ul BSA-1mg/ml, 5ul ATP-10mM -2ul dNTPs 10 mM), 5 ul T4 PNK enzyme, 5 µl T4 polymerase (3 units) to each well. Samples were incubated in a thermal cycler at 12C for 15 min, 25C for 15 min, and finally cooled to 4C. The SPRI bead clean up method was used to purify the product (147 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40 µl EB). The A-base addition was performed by adding 20 µl master mix (17 µl A-base add mix, 3 µl Klenow (3’->5’ exonuclease) to each well and incubated at 37C for 30 min. in a thermal cycler. SPRI bead clean up method was used to purify the product (132 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 19 µl EB). Adaptor ligation was performed by adding 34 µl of a master mix (29 µl 2x DNA ligase buffer, 5 µl DNA ligase) to each well. Finally 5 µl PE Indexed oligo adaptors (0.75 uM ) was added to each well and samples were incubated 25C for 15 min in a thermal cycler. SPRI bead clean up with size selection was used to purify the ligated products (15.5 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40 µl EB). Finally, enrichment PCR was performed by adding 10 µl of a master mix (2 µl Forward/Reverse Index Primer, 0.5 µl dNTP mix, 5 µl 10x Pfu Ultra Buffer, 1 µl Pfu Ultra-II Fusion, 1.5 µl Nuclease free water) to each well. Plate was transferred to a thermal cycler and ran a Pfu amplification program at 95C for 2 min, 16 cycles of: 95C for 30 sec, 55C for 30 sec, 72C for 60 sec, and finally 72C for 10 min. The final SPRI clean up coupled to size selection was performed (35 µl SPRI beads was added to each sample and eluted in 40 µl).
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
 
Data processing We used Illumina bcl2fastq version 2.17.1.14 software to generate fastq files.
reads were aligned to mouse mm10 reference genome using bowtie2 software, with default parameters.
for transcription factors, peaks were identified with MACS version 1.4.2-157. Whole-cell extract was used as control to define a background model. Duplicate reads aligned to the exact same location are excluded by MACS default configuration.
Chromatin modification coverage in the promoters was calculated using in-house script. Shortly, promoters were defined as 1Kb region around the TSS, based on mm10 assembly. Each promoter was divided to 50bp size bins, and the coverage in each bin is estimated.
In the proccessed matrix file, for each promoter is the coverage in 20 bins, 50bp each.
Genome_build: mm10
 
Submission date Aug 10, 2017
Last update date Jul 16, 2018
Contact name Noa Novershtern
E-mail(s) noa.novershtern@weizmann.ac.il
Organization name Weizmann Institute of Science
Department Molecular Genetics
Street address Weizmann Institute
City Rehovot
ZIP/Postal code 7610001
Country Israel
 
Platform ID GPL17021
Series (2)
GSE102517 High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems [ChIP-Seq]
GSE102518 High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems
Relations
BioSample SAMN07495498
SRA SRX3087077

Supplementary file Size Download File type/resource
GSM2739969_H3K4me3_Day2.prom_500.matrix.txt.gz 2.8 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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