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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 12, 2015 |
| Title |
H3K56ac ChIP-Seq Sirt6 Sirt6 KO RA Treated |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells (ESCs)
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| Organism |
Mus musculus |
| Characteristics |
genotype/variation: Sirt6 KO
treatment: RA Treated
chip antibody: H3K56ac (abcam, ab76307)
strain: 129
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| Treatment protocol |
ChIP-Seq experiments used either untreated mouse ESCs or retinoic acid (RA) differentiated mouse ESCs. The mouse ESC differentiation protocol used RA was as follows: ESCs were grown in LIF(-) medium [DMEM + GlutMax TM-1 (invitrogen) medium + 15% Serum (w/o LIF)]. On the next day, the media was changed to RA-containing LIF(-) medium [DMEM + GlutMax TM-1 (invitrogen) medium + 15% Serum (w/o LIF) + retinoic acid (RA) at dilution 1:10000/10cm plate]. Two days later, the media was changed back to LIF(-) medium [DMEM + GlutMax TM-1 (invitrogen) medium + 15% Serum (w/o LIF)]. Cells were harvested the day after and processed for ChIP seq analysis as described above. The RA stock used 0.01 M All trans-RETINOIC ACID (Sigma Product number: R 2625) in DMSO (10000x solution) used following the instructions from manufacturer.
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| Growth protocol |
Mouse ESCs (mESCs) derived from Sirt6 Sirt6 KO and WT 129 mouse strain were maintained on gamma-irradiated mouse embryonic fibroblasts (MEFs) in knocSirt6 KOut DMEM medium (GIBCO) containing 15% ES-qualified FBS, 0.1 mM each of nonessential amino acids, 2 mM L-glutamine, 0.1 mM beta-mercaptoethanol, 50 units/ml penicillin/streptomycin (Invitrogen) and supplemented with leukemia inhibiting factor (LIF). For all experiments described, cells were trypsinized and plated for 30 min on standard tissue culture dishes to remove feeder cells before floating ES cells were collected and re-plated on gelatin-coated dishes or wells prior differentiation towards EBs.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation (ChIP) steps were performed using a modified version of our previous protocols (e.g., Ram & Goren et. al. (2011) Combinatorial Patterning of Chromatin Regulators Uncovered by Genome-wide Location Analysis in Human Cells. Cell. 147 (7). and Garber & Yosef et. al. (2012) A High-Throughput Chromatin Immunoprecipitation Approach Reveals Principles of Dynamic Gene Regulation in Mammals. Molecular Cell 47 (5).) adapted to the Bravo liquid handling platform (Agilent). Cells were crosslinked using formaldehyde (1%, 37oC for 10 min), and then quenched with glycine (5min at 37oC). The fixed cells were lysed in Lysis buffer (1% SDS, 10mM EDTA and 50mM Tris-HCl pH 8.1, containing protease inhibitors (Roche, 04693159001). Chromatin was sheared using Covaris E-220 at 4oC to a size range between 200 and 800 bp. 1 to 5µg of antibody were incubated 2 hours with a mix of Protein-A and Protein-G Dynabeads (Invitrogen, 100-02D and100-07D, respectively) in blocking buffer (PBS supplemented with 0.5% TWEEN and 0.5% BSA). Beads were washed added to the chromatin lysate, and incubated overnight. Next, samples were washed 6 times with RIPA buffer, twice with RIPA buffer containing 500mM NaCl, twice with LiCl buffer (10mM TE, 250mM LiCl, 0.5% NP-40, 0.5% DOC), twice with TE (10Mm Tris-HCl pH 8.0, 1mM EDTA), and then eluted in ChIP-Elution Buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris Hcl pH 8.0) at 65oC, 4 hours, and then treated with RNaseA (Roche, 11119915001) for 30 min and Proteinase K (NEB, P8102S) for 2 hours.
Illumina Library construction reactions were performed as we described previously (Garber & Yosef et. al. 2012). SPRI (Solid-phase reversible immobilization; AMPure XP beads (Agencourt)) cleanup steps were done using the Bravo liquid handling platform (Agilent) and a modified version of (Fisher et al., 2011). After the final ChIP step, 120µl SPRI was added to the reverse-crosslinked samples, pipette-mixed 15 times and incubated for 2 minutes. Supernatant was separated from the beads using a 96-well magnet for 4 minutes. Beads were washed on the magnet with 70% ethanol and then air dried for 4 minutes. DNA was eluted in 40µl EB buffer (10 mM Tris-HCl pH 8.0) by pipette mixing (25 times). The remainder of the library construction process (DNA end-repair, A-base addition, adaptor ligation and enrichment) the SPRI cleanup involves addition of PEG buffer (20% PEG and 2.5 M NaCl) to the reaction products. Next, plates were transferred to a magnet plate, incubated for 4 minutes and supernatant removed. Beads are washed on the magnet with 150µl 70% ethanol and air dried for 4 minutes. The DNA is eluted with 40µl of EB buffer (mixing 25 times). Reagent kits are prepared in advance for all enzymatic steps (New England Biolabs). The DNA end-repair was performed by adding 27µl of a master mix (17µl master mix (5µl T4 buffer, 5µl BSA-1mg/ml, 5µl ATP-10mM -2µl dNTPs 10 mM)) plus 5µl T4 PNK enzyme, 5µl T4 polymerase (3 units) to each well. Samples were incubated in a thermal cycler at 12oC for 15 min, 25 oC for 15 min, and finally cooled to 4 oC. The SPRI bead clean up method was used to purify the product (147µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40µl EB). The A-base addition was performed by adding 20µl master mix (17µl A-base add mix, 3µl Klenow to each well and incubated at 37oC for 30 min in a thermal cycler. Product was purified using 132µl PEG buffer and eluted in 19µl EB. Adaptor ligation: was performed by adding 34µl of a master mix (29µl 2x DNA ligase buffer, 5µl DNA ligase) and 5µl indexed oligo adaptors (0.75 uM ) followed by 15min incubation in 25oC in a thermal cycler. Next, ligated products were purified using SPRI bead clean up with size selection (15.5µl of PEG buffer) and eluted in 40µl EB. The final step of PCR enrichment was was performed by adding 10µl of a master mix (2µl Forward/Reverse Index Primer, 0.5µl dNTP mix, 5µl 10x PfuUltra Buffer, 1µl PfuUltra-II Fusion, 1.5µl Nuclease free water) to each well. Using a thermal cycler, programed to 95C for 2 min, followed by 16 cycles of (95oC for 30 sec, 55oC for 30 sec, 72oC for 60 sec), and a final incubation at 72oC for 10 min. The last SPRI clean up was coupled to size selection (35µl SPRI beads were added to each sample and eluted in 40µl).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
H3K56ac ChIP-Seq in Sirt6 Sirt6 KO mESC after differentiation with RA Treatment
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| Data processing |
ChIP libraries were indexed, pooled, and sequenced on Illumina HiSeq-2500 sequencers at the Broad Institute sequencing center.
ChIP-seq reads were aligned to the mouse mm9 genome assembly using bwa version 0.5.9 with parameters '-q 5 -l 32 -k 2 -t 4 -o 1 -f' for the aln command and '-t 4 -T -P -f' for the sampe command.
ChIP-seq duplicate reads were marked with Picard Tools MarkDuplicates version number 1.764.
Peak calling with MACS2 version 2.0.10 and parameters '-g mm -f BAM -B -q 0.01'
Ran the IGVtools version 2.3.18 utility 'count' on aligned bam files to compute the average alignment over 25 base windows across the genome and generate binary tiled data .tdf files.
Genome_build: mm9
Supplementary_files_format_and_content: txt files are ChIP-Seq peak summary files from MACS2 in a tab delimited text file format.
Supplementary_files_format_and_content: tdf files are binary tiled data files of aligned ChIP-Seq data from the IGVtools count utility that conatin average alignment counts across the genome and can viewed in the IGV genome browser.
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| Submission date |
Feb 10, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Kenneth N Ross |
| E-mail(s) |
Kenneth.Ross@dfci.harvard.edu
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| Organization name |
Dana-Farber Cancer Institute
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| Department |
Pediatric Oncology
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| Street address |
450 Brookline Ave., Rm M640
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE65836 |
The Histone Deacetylase Sirt6 Controls Embryonic Stem Cell Fate Via Tet-Mediated Production of 5-Hydroxymethylcytosine |
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| Relations |
| BioSample |
SAMN03339764 |
| SRA |
SRX873352 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1607378_2_JP4-H3K56ac_HA61CADXX.2.tdf |
194.8 Mb |
(ftp)(http) |
TDF |
| GSM1607378_JP4_Sirt6_KO_RA_H3K56ac_140828_peaks.txt.gz |
65.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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