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Sample GSM515676 Query DataSets for GSM515676
Status Public on Apr 02, 2010
Title PolII_Nterm_Doxycycline0hr_ChIP_Seq
Sample type SRA
 
Source name ZHBTc4 mES cells
Organism Mus musculus
Characteristics cell type: ZHBTc4 mES cells
shrna: no
small molecule: Doxycycline 0 hour
chip antibody: RNA Pol II (Rpb1 N-terminus)
Treatment protocol For location analysis following Spt5 and NelfA knockdown, shRNA plasmids targeting the mouse Spt5 and NelfA mRNAs and an empty plasmid (control) (Open Biosystems, Huntsville, AL RMM4534-NM_013676, RMM4534-NM_011914, and RMM4534_NM_010849, RHS4080) were used. We purchased and tested each set of shRNA hairpins for ability of each hairpin to knockdown the mRNA of the factor of interest. We then selected the hairpin that performed the best for use in ChIP-seq analysis. For Spt5, hairpin TRCN0000092761 was used. For NelfA, hairpin TRCN0000124874 was used. For c-Myc, hairpin TRCN0000042516 was used. 293T cells were plated in 6-well dishes at 6x10^5 cells/well. The shRNA plasmids and lentiviral components were co-transfected into 293T cells. V6.5 mES cells were plated in T-75 flasks at 2x10^6 cells/flask. Viral media was collected 48 hours after co-transfection and the V6.5 mES cells were directly infected with the viral media 24 hours after initial plating of the mES cells. The infection media was 1:2 viral media:mES cell media with 2mM polybrene. The efficiently infected cells were selected for 24 hours post infection with mES cell media containing 2μM puromycin. V6.5 cells were cross-linked 72 hours post selection and frozen for ChIP-Seq experiments and western blotting.
For location analysis on mES cells following treatment with small molecule inhibitors, cells were grown two passages off feeders and prior to formaldehyde crosslinking, the cells were treated by addition of the indicated final concentration of flavopiridol (1μM for 1 hour for ChIP-chip and ChIP-seq experiments, or the indicated concentration and time for Western blot analysis), or c-Myc/Max inhibitor 10058-F4 (50μM for 6 hours for ChIP-seq experiments or the indicated time for Western blot analysis), both dissolved in DMSO, to the growth medium. 50μM is within the concentration range commonly used for 10058-F4 in vivo to investigate c-Myc function (Arabi et al., 2005; Fang et al., 2008; Faumont et al., 2009; Follis et al., 2008; Hammoudeh et al., 2009; Khanna et al., 2009; Lee et al., 2009; Sampson et al., 2007; Wang et al., 2007). As a control, vehicle alone (DMSO) was added to the growth medium at the same final volume as with drug. Small molecule inhibitors used were: Flavopiridol (Sigma cat #F3055), and c-Myc inhibitor 10058-F4 (Sigma cat #F3680).
For location analysis following Oct4 shutdown, ZHBTc4 mES cells (Niwa et al., 2000) were grown under standard mES cell culture conditions and expanded for two passages off MEF feeders. ES cell culture media with 2 μg/ml doxycycline was added to the cells for 0 hours, 12 hours and 24 hours prior to formaldehyde crosslinking. Loss of Oct4 was assessed using Western Blot analysis.
Growth protocol V6.5 cells were grown under standard mES cell conditions as described previously (Boyer et al., 2005). Briefly, cells were grown on 0.2% gelatinized tissue culture plates in ESC media; DMEM-KO supplemented with 15% fetal bovine serum, 1000 U/mL LIF, 100 uM nonessential amino acids, 2 mM L-glutamine, 100 U/mL penicillin, 100 ug/mL streptomycin and 8 nL/mL of 2-mercaptoethanol
Extracted molecule genomic DNA
Extraction protocol ChIP was done following the Agilent Mammalian ChIP-on-chip protocol (version 9.1, Nov 2006). In summary, mES cells were grown as described above and cross-linked for 15 minutes at room temperature by the addition of one-tenth of the volume of 11% formaldehyde solution (11% formaldehyde, 50mM Hepes pH7.3, 100mM NaCl, 1mM EDTA pH8.0, 0.5mM EGTA pH8.0) to the growth media followed by two washes with PBS. Cells were scraped and frozen in liquid nitrogen. 100ul of Dynal magnetic beads (Sigma) were blocked with 0.5% BSA (w/v) in PBS. Magnetic beads were bound with 10ug of the indicated antibody. Antibodies used are as follows: Pol II (all; Rpb1 N-terminus): Santa Cruz sc-899; Ser5P Pol II: Abcam ab5131; Ser2P Pol II: Abcam (H5 clone) ab24758 with Upstate IgG-IgM linker antibody 12-488; Spt5: gift from Yuki Yamaguchi and Hiroshi Handa (Wada et al., 1998); NelfA: Santa Cruz (A-20) sc-23599;; Ctr9: Bethyl labs A301-395; and Max: Santa Cruz sc-197. For all of the experiments analyzing Pol II occupancy following shRNA-mediated knockdown, flavopiridol, or 10058-F4 treatment, the Pol II (all; Santa Cruz sc-899, Pol II N-20) antibody was used. Crosslinked cells were lysed with lysis buffer 1 (50mM Hepes pH7.3, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100) and washed with lysis buffer 2 (10mM Tris-HCl pH8.0, 200mM NaCl, 1mM EDTA pH8.0 and 0.5mM EGTA pH8.0). For Spt5 ChIPs, cells were resuspended and sonicated in lysis buffer 3 (10mM Tris-HCl pH8.0, 100mM NaCl, 1mM EDTA pH8.0, 0.5mM EGTA pH8.0, 0.1% Na-Deoxycholate and 0.5% N-lauroylsarcosine) for 8 cycles at 30 seconds each on ice (18 watts) with 60 seconds on ice between cycles. Triton X-100 was added to a final concentration of 1% to the sonicated lysates. Sonicated lysates were cleared and incubated overnight at 4oC with magnetic beads bound with antibody to enrich for DNA fragments bound by the indicated factor. Beads were washed four times with RIPA (50mM Hepes pH7.3, 500mM LiCl, 1mM EDTA, 1% NP-40 and 0.7% Na-Deoxycholate) and once with TE + 50mM NaCl. Bound complexes were eluted in elution buffer (50mM Tris-HCl pH8.0, 10mM EDTA pH8.0, 1% SDS) at 65oC for 15 minutes with occasional vortexing. Cross-links were reversed overnight at 65oC. RNA and protein were digested using RNAse A and Proteinase K, respectively and DNA was purified with phenol chloroform extraction and ethanol precipitation. For Pol II Ser5P, Pol II (all), NelfA, Ctr9 and Max ChIPs, cells were resuspended and sonicated in sonication buffer (50mM Tris-HCl pH7.5, 140mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS) for 8 cycles at 30 seconds each on ice (18 watts) with 60 seconds on ice between cycles. Sonicated lysates were cleared and incubated overnight at 4oC with magnetic beads bound with antibody to enrich for DNA fragments bound by the indicated factor. Beads were washed three times with sonication buffer, one time with sonication buffer with 500mM NaCl, one time with LiCl wash buffer (20mM Tris pH8.0, 1mM EDTA, 250mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate) and one time with TE. DNA was eluted in elution buffer. Cross-links were reversed overnight. RNA and protein were digested using RNAse A and Proteinase K, respectively and DNA was purified with phenol chloroform extraction and ethanol precipitation. For Pol II Ser2P ChIP, cells were resuspended and sonicated in sonication buffer II (50mM Tris-HCl pH7.5, 140mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS) for 8 cycles at 30 seconds each on ice, at 18 watts with 60 seconds on ice between cycles. Sonicated lysates were cleared and incubated overnight at 4oC with magnetic beads bound with antibody to enrich for DNA fragments bound by Pol II Ser2P. Beads were washed two times with sonication buffer II, one time with LiCl wash buffer (2mM Tris pH8.0, 0.02mM EDTA, 50mM LiCl, 0.1% NP-40, 0.1% Na-deoxycholate) and one wash with TE. DNA was eluted in elution buffer. This protocol is similar to that used in (Stock et al., 2007). Cross-links were reversed overnight. RNA and protein were digested using RNAse A and Proteinase K, respectively and DNA was purified with phenol chloroform extraction and ethanol precipitation. DNA was prepared for sequencing according to a modified version of the Illumina/Solexa Genomic DNA protocol. Fragmented DNA was prepared for ligation of Solexa linkers by repairing the ends and adding a single adenine nucleotide overhang to allow for directional ligation. A 1:100 dilution of the Adaptor Oligo Mix (Illumina) was used in the ligation step. A subsequent PCR step with limited (18) amplification cycles added additional linker sequence to the fragments to prepare them for annealing to the Genome Analyzer flow-cell. After amplification, a narrow range of fragment sizes was selected by separation on a 2% agarose gel and excision of a band between 150-300 bp (representing shear fragments between 50 and 200nt in length and ~100bp of primer sequence). The DNA was purified from the agarose and diluted to 10 nM for loading on the flow cell. The DNA library (2-4 pM) was applied to the flow-cell (8 samples per flow-cell) using the Cluster Station device from Illumina. The concentration of library applied to the flow-cell was calibrated such that polonies generated in the bridge amplification step originate from single strands of DNA. Multiple rounds of amplification reagents were flowed across the cell in the bridge amplification step to generate polonies of approximately 1,000 strands in 1μm diameter spots. Double stranded polonies were visually checked for density and morphology by staining with a 1:5000 dilution of SYBR Green I (Invitrogen) and visualizing with a microscope under fluorescent illumination. Validated flow-cells were stored at 4 degrees C until sequencing. Flow-cells were removed from storage and subjected to linearization and annealing of sequencing primer on the Cluster Station. Primed flow-cells were loaded into the Illumina Genome Analyzer 1G. After the first base was incorporated in the Sequencing-by-Synthesis reaction the process was paused for a key quality control checkpoint. A small section of each lane was imaged and the average intensity value for all four bases was compared to minimum thresholds. Flow-cells with low first base intensities were re-primed and if signal was not recovered the flow-cell was aborted. Flow-cells with signal intensities meeting the minimum thresholds were resumed and sequenced for 26 or 32 cycles.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer
 
Description Chromatin IP against total Pol II in cells treated with doxycycline for 0 hours
Data processing Images acquired from the Illumina/Solexa sequencer were processed through the bundled Solexa image extraction pipeline which identified polony positions, performed base-calling and generated QC statistics. Sequences were aligned using ELAND software to NCBI Build 36 (UCSC mm8) of the mouse genome (WIG files linked to GSE20530). Only sequences that mapped uniquely to the genome with zero or one mismatch were used for further analysis. When multiple reads mapped to the same genomic position, a maximum of two reads mapping to the same position were used. Sequence reads from multiple flow cells for each IP target were combined. Each read was extended 100bp, towards the interior of the sequenced fragment, based on the strand of the alignment. Across the genome, in 25 bp bins, the number of ChIP-Seq reads within a 1kb window surrounding each bin (+/- 500bp) was tabulated. The 25bp genomic bins that contained statistically significant ChIP-Seq enrichment were identified by comparison to a Poissonian background model. Assuming background reads are spread randomly throughout the genome, the probability of observing a given number of reads in a 1kb window can be modeled as a Poisson process in which the expectation can be estimated as the number of mapped reads multiplied by the number of bins (40) into which each read maps, divided by the total number of bins available (we estimated 70%). Enriched bins within 1kb of one another were combined into regions. The complete set of RefSeq genes was downloaded from the UCSC table browser (http://genome.ucsc.edu/cgi-bin/hgTables?command=start) on December 20, 2008. Gene with enriched regions within 5kb of their transcription start site were called bound. The Poissonian background model assumes a random distribution of background reads, however we have observed significant deviations from this expectation. Some of these non-random events can be detected as sites of apparent enrichment in negative control DNA samples and can create many false positives in ChIP-Seq experiments. To remove these regions, we compared genomic bins and regions that meet the statistical threshold for enrichment to a set of reads obtained from Solexa sequencing of DNA from whole cell extract (WCE) in mES cells (WCE sample from Cole et al. 2008). We required that enriched bins and enriched regions have five-fold greater ChIP-Seq density in the specific IP sample, compared with the control sample, normalized to the total number of reads in each dataset. This served to filter out genomic regions that are biased to having a greater than expected background density of ChIP-Seq reads.
 
Submission date Feb 25, 2010
Last update date May 15, 2019
Contact name Charles Lin
E-mail(s) chazlin@mit.edu
Phone 6172585218
Fax 6172580376
URL http://web.wi.mit.edu/young/
Organization name Massachusetts Institute of Technology
Department Biology
Lab Richard A. Young
Street address 9 Cambridge Center
City Cambridge
State/province MA
ZIP/Postal code 02142
Country USA
 
Platform ID GPL9185
Series (2)
GSE20485 Promoter proximal pausing and its regulation by c-Myc in embryonic stem cells
GSE20530 Promoter proximal pausing and its regulation by c-Myc in embryonic stem cells: ChIP-Seq
Relations
SRA SRX017070
BioSample SAMN00009364

Supplementary data files not provided
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Processed data are available on Series record
Raw data are available in SRA

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