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| Status |
Public on Nov 20, 2013 |
| Title |
Hdac6KD_Tip60ChIP |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: E14
genotype/variation: Tip60-H3F/ Hdac6KD
chip antibody: Flag-M2 (Sigma, F1804)
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| Treatment protocol |
In one 10 cm2 dish, 1X10^6 cells were infected with 30 ul of concentrated lentivirus. After 12-15 hours, the media was replaced and cells were cultured until 72 hours post-infection.
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| Growth protocol |
Feeder-free growth on gelatin coated dishes in standatd ES medium: KO DMEM + 10% FBS, glutamine, LIF, non-essential amino acids, beta-mercaptoethanol.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells from ~80 % confluent 10 cm dishes were crosslinked by adding fixation solution (1% formaldehyde, 0.1M NaCl, 1mM EDTA, 50mM HEPES⋅KOH pH 7.6) for 10 minutes at room temperature. Crosslinking was quenched with 125mM Glycine for 5 minutes. Cells were washed twice with cold PBS containing protease inhibitors (Roche), and pelleted at 1000g for 5 minutes at 4°C. Cell pellets were either flash frozen in liquid nitrogen and stored at -80°C or immediately sonicated. Pellets were resuspended in Lysis buffer 1 (50mM HEPES⋅KOH pH 7.6, 140mM NaCl, 1mM EDTA, 10% (v/v) Glycerol, 0.5% NP-40, 0.25% Triton X-100) including protease inhibitors and incubated for 10 minutes at 4°C. After centrifugation at 1350g for 5 minutes, pellets were resuspended in Lysis buffer 2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) containing protease inhibitors and incubated for another 10 minutes at 4°C. Pellets were collected after centrifugation at 1350g for 5 minutes and resuspended in Lysis buffer 2 for sonication. Samples were transferred into 15 ml tubes (Falcon) and sonicated in a Bioruptor set to high for 2 cycles (10 minutes for one cycle with 30 sec on/ 30 sec off). The supernatants were collected after a 13000 rpm spin for 10 minutes at 4°C. 50μl Protein G Magnetic beads (NEB) were washed twice with PBS with 5mg/ml BSA and 10μg of anti-Flag M2 antibody (Sigma) coupled in 500μl PBS with 5mg/ml BSA overnight at 4°C. Immunoprecipitation was performed with antibody-coupled beads and sonicated supernatants in ChIP buffer (20mM Tris-HCl pH8.0, 150mM NaCl, 2mM EDTA, 1% Triton X-100) overnight at 4°C. Magnetic beads were washed twice with ChIP buffer, once with ChIP buffer including 500mM NaCl, 4 times with RIPA buffer (10mM Tris-HCl pH8.0, 0.25M LiCl, 1mM EDTA, 0.5% NP-40, 0.5% Na⋅Deoxycholate), and once with TE buffer (pH 8.0). Chromatin was eluted twice from washed beads by adding elution buffer (20mM Tris-HCl pH8.0, 100mM NaCl, 20mM EDTA, 1% SDS) and incubating for 15 minutes at 65°C. The crosslinking was reversed at 65°C for 6hr and RNase A (Sigma) was added for 1hr at 37°C followed by proteinase K (Ambion) treatment overnight at 50°C. ChIP-enriched DNA was purified using Phenol/Chloroform/Isoamyl alcohol extractions in phase-lock tubes.
Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (exo minus) and dATP to yield a protruding 3- 'A' base for ligation of barcoded adapters which have a single 'T' base overhang at the 3’ end. DNA purification on Zymo Research PCR purification columns (Zymo, Irvine, CA) was performed following each enzyme reaction. The adaptor-ligated material was then PCR amplified with Phusion polymerase using 16 cycles of PCR before size selection of 200-300 bp fragments on a 2% agarose gel. Libraries with different barcodes were pooled together and single-end sequencing (50 bp) was performed on an Illumina HiSeq2000.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Basecalls performed using CASAVA version 1.8.2
Barcode sequences were trimmed and ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie allowing at most one mismatch in every alignment.
For multimappers, only one alignment was chosen randomly by the -M 1 parameter setting.
Each aligned location was extended downstream to a length of 150 bp. Any extension that exceeded the end of the chromosome was clipped. The extended mapped locations overlapping with simple repeats annotated by RepBase were removed.
For each remaining read along with its occurrence, we calculated the relative distance to the nearest TSS and for each TSS tallied the sum of read occurrence from its upstream 2000 bp to downstream 2000 bp. The occurrences were normalized and binned in 20 bp intervals.
Genome_build: mm9
Supplementary_files_format_and_content: The aggregation signals around each TSS (+/- 2000bp) are tabulated in flat format (*.txt). The occurrences were normalized and binned in 20 bp intervals.
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| Submission date |
Nov 09, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Benson Chen |
| E-mail(s) |
b5chen@ucsd.edu
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| Organization name |
UC San Diego
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| Department |
Cellular and Molecular Medicine
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| Lab |
Bing Ren
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| Street address |
9500 Gilman Dr. CMM East, Room 2071
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093-0653 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE42209 |
Hdac6 regulates Tip60-p400 function in stem cells (sequencing) |
| GSE42329 |
Hdac6 regulates Tip60-p400 function in stem cells |
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| Relations |
| SRA |
SRX204802 |
| BioSample |
SAMN01814646 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1035415_Hdac6KD_Tip60IP.raw.txt.gz |
3.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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