cell type: V6.5 ES cells shrna: no small molecule: Flavopiridol 1 hour chip antibody: none
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. 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 2mM 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), dissolved in DMSO, to the growth medium. As a control, vehicle alone (DMSO) was added to the growth medium at the same final volume as with drug. Flavopiridol (Sigma cat #F3055) was purchased from Sigma.
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: Spt5: gift from Yuki Yamaguchi and Hiroshi Handa (Wada et al., 1998); and NelfA: Santa Cruz (A-20) sc-23599). 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 NelfA 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.
Label
Cy3
Label protocol
Purified immunoprecipitated DNA was amplified using two rounds of ligation mediated PCR (LM PCR), as described in (Lee et al., 2006b) and the Agilent Mammalian ChIP-on-chip protocol (version 9.1, Nov 2006). LM-PCR immunoprecipitated DNA was labeled with Cy5, LM-PCR input DNA was labeled with Cy3 using Invitrogen Bioprime random primer labeling kit.
cell type: V6.5 ES cells shrna: no small molecule: Flavopiridol 1 hour chip antibody: NelfA
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. 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 2mM 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), dissolved in DMSO, to the growth medium. As a control, vehicle alone (DMSO) was added to the growth medium at the same final volume as with drug. Flavopiridol (Sigma cat #F3055) was purchased from Sigma.
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: Spt5: gift from Yuki Yamaguchi and Hiroshi Handa (Wada et al., 1998); and NelfA: Santa Cruz (A-20) sc-23599). 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 NelfA 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.
Label
Cy5
Label protocol
Purified immunoprecipitated DNA was amplified using two rounds of ligation mediated PCR (LM PCR), as described in (Lee et al., 2006b) and the Agilent Mammalian ChIP-on-chip protocol (version 9.1, Nov 2006). LM-PCR immunoprecipitated DNA was labeled with Cy5, LM-PCR input DNA was labeled with Cy3 using Invitrogen Bioprime random primer labeling kit.
Hybridization protocol
Agilent mouse 4x44k custom arrays were hybridized according to the Agilent Mammalian ChIP-on-chip protocol (version 9.1, Nov 2006) was followed. In brief, mouse Cot1 DNA, Agilent blocking buffer (1X final conc.), Agilent hybridization buffer (1X final conc.), was added to Cy5- and Cy3-labeled DNA. The mixture was incubated at 95oC for 3 minutes, followed by 37oC for 30 minutes. Sample was centrifuged for 1 min and sample was hybridized to Agilent DNA microarray. For experiments testing effects of flavopiridol on NelfA and Spt5 occupancy, and NelfA or Spt5 occupancy following Spt5 or NelfA knockdown, respectively, ChIP samples were hybridized to Agilent arrays MTvB (44,000 features covering the promoter region, from approximately -6kb to +2kb, of approximately 10% of mouse genes – MTvB). DNA was hybridized to microarray for 40 hours at 65oC.
Scan protocol
Microarray was washed and scanned following the Agilent Mammalian ChIP-on-chip protocol. The Agilent DNA microarray scanner BA was used. PMT settings were set manually to normalize bulk signal in the Cy3 and Cy5 channel.
Description
AFE_030909_251520410225_S02_1_4_NelfA_mES_flavo
Data processing
Array images were quantified and statistical significance of differential expression for each hybridization was calculated using Agilent’s Feature Extraction Image Analysis software with the default two-color Young lab protocol. Data was processed as described in (Lee et al., 2006a) to calculate enrichment ratios and determine bound regions.
