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| Status |
Public on Mar 08, 2012 |
| Title |
shluc.Input |
| Sample type |
SRA |
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| Source name |
Input control
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| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
shRNA construct: luciferase (shluc)
chip antibody: none (input)
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| Treatment protocol |
Cells were depleted of ATRX using stably integrated lentiviral construct expressing shRNA constructs (Open Biosystems) targeting ATRX RNA for degradation (TRCN0000013592). Cells bearing lentiviral constucts targeting luciferase for degradation were used as the control. Cells were maintained under puromycin selection (2ug/ml) after being infected with virus (spin procotol).
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| Growth protocol |
Cells were grown in 75cm^2 suspension culture flasks (RPMI media) and maintained under puromycin selection (2ug/ml)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
mH2A1 ChIP (ab37264, ABCAM), was preformed with 100ug of MNase digested chromatin as previously described (Hori et al. 2008). Total of 25ng MNase-ChIPed DNA was processed for library preparation from each sample. All enzymes used are from NEB unless stated otherwise. 1. DNA was End Repaired in 50ul reaction containing 40ul of DNA, 5ul T4 DNA ligase buffer with 10mM ATP, 2ul 10mM dNTP, 1ul Klenow DNA pol (1u/ul), 1ul T4 DNA pol and 1ul T4 PNK. The reaction was incubated for 30min at 20C. Products were purified in Qiagen PCR purification column in 35ul. 2. Adding over-hang A to the 3’ end in 50ul reaction containing 34ul DNA, 5ul 10X NEB buffer 2, 10ul 1mM dATP and 1ul Klenow exo-. The reaction was incubated for 30min at 37C. Products were purified in Qiagen PCR Mini elute purification column in 14ul. 3. Adapter ligation in 30ul reaction containing 13ul DNA, 15ul 2X T4 DNA ligase buffer, 1ul of 1:200 dilution of Illumina true seq DNA sample prep kit and 1ul quick T4 DNA ligase. The reaction was incubated for 15min at 25C. Products were purified in Qiagen Mini elute PCR purification column in 15ul. 4. Size selection. Ligated products were loaded onto 2% Agarose gel (1XTAE), and run for 1 hour at 120V. DNA was viewed on low-wave trans illuminator and size selected at the 300-400bp range using the Kb+ invitrogen DNA ladder. Products were purified in Qiagen PCR purification column in 25ul. 5. PCR amplification was done in 50ul reaction containing 25ul 2X master mix (2X Phusion HF, FINZYMES), 2ul PCR primers (illumina) and 23ul DNA. PCR program according to the illumina protocol. Products were purified in Qiagen Mini elute PCR purification column in 15ul. PCR products were then run on an Agilent DNA 1000 chip for quantification and submitted for sequencing on the Hi-Seq 2000 illumina platform. Sh92 and shluc were multiplexed. We obtained a total of 67,219,237 reads for sh92, 56,540,184 for shluc and 148,165,330 for Input
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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 |
100bp reads were aligned to the hg19 genome assembly (NCBI build 37) using Bowtie short read aligner (v 0.12.7) (Langmead et al. 2009), with the following parameters: seed of 50bp, maximum 2 mismatches, suppression (m) = 20, and reported alignments (k) = 20. Aligned reads were then converted to BED files (sh92.mH2A-ChIP.BED, shluc.mH2A-ChIP.BED and shluc.Input.BED) Wiggle files (HAFEZ, unpublished pipeline D.H.), were generated using a 500bp window sliding 250bp, counting the number of aligned reads (5' end of each aligned read), for both ChIP and Input samples. The number of alignments from each window was normalized to the total number of alignments and scaled by factor of 10^7, to allow comparison between different samples (sh92.mH2A-ChIP.wig, shluc.mH2A-ChIP.wig and shluc.Input.wig). The MACS software (v 1.4.1) (4) was used to identify peaks (pvalue cutoff = 1.00e-04, shift size = 75, max duplicate reads allowed = 2, (nomodel), fragment size = 100 -t) (sh92.mH2A-ChIP.peaks.BED, shluc.mH2A-ChIP.peaks.BED) (Zhang et al. 2008).
Genome Build:
shluc.Input.BED: hg19 (NCBI Build37)
shluc.Input.wig: hg19 (NCBI Build37)
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| Submission date |
Jan 25, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Emily Bernstein |
| E-mail(s) |
emily.bernstein@mssm.edu
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| Phone |
(212) 824-9335
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| Organization name |
Mount Sinai School of Medicine
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| Department |
Oncological Sciences
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| Lab |
Bernstein Lab
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| Street address |
1470 Madison Avenue, 6th floor Rm 302
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE35339 |
ATRX-mediated chromatin association of histone variant macroH2A1 regulates alpha globin gene expression |
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| Relations |
| SRA |
SRX117840 |
| BioSample |
SAMN00779068 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM866352_shluc.Input.BED.gz |
2.1 Gb |
(ftp)(http) |
BED |
| GSM866352_shluc.Input.wig.gz |
40.3 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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