cell type: Epstein-Barr-Virus (EBV)-transformed human lymphoblastoid B cell line LCL 721 antibody: none
Treatment protocol
standard growth conditions, no treatment
Growth protocol
LCL 721 cells were cultured for five passages (Px5) in RPMI 1640 medium at 37°C and 5% CO2 in a humidified incubator. Medium was supplemented with 10% heat-inactivated foetal bovine serum, 5 mM L-glutamine and 100 units/ml penicillin-streptomycin.
Extracted molecule
genomic DNA
Extraction protocol
1x10E8 cells (cell density 0.4x10E6 cells/ml) were pelleted by centrifugation (1200 rpm, 5 min), washed with PBS, and the pellet was resuspended in 36 ml of PBS (all steps at room temperature). Cells were fixed by adding 4 ml of a freshly prepared 10% formaldehyde solution (10% formaldehyde, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes-KOH pH 8.0). Cross-linking was done for 9 min at room temperature, followed by quenching with 125 mM glycine, with immediate transfer of the cell suspension to and 5 min incubation on ice. Cells were washed twice with ice-cold PBS and sequentially lysed by resuspending the cell pellet in 5 ml of ice-cold ChIP lysis buffer 1 (50 mM Hepes-KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100, freshly added 1x protease inhibitor cocktail) and 10 min rotation at 4°C. Cells were collected by centrifugation (4000 rpm, 10 min, 4°C), followed by resuspension in 5 ml of ice-cold ChIP lysis buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, freshly added 1x protease inhibitor cocktail) and 10 min rotation at 4°C. After centrifugation (4000 rpm, 10 min, 4°C), the pellet was resuspended in 3 ml of ice-cold ChIP lysis buffer 3 (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, freshly added 0.5% N-lauryl sarcosine, 0.1% sodium deoxycholate and 1x protease inhibitor cocktail). Acid-washed glass beads (212-300 microns) were added, and the crosslinked chromatin was sheared to an average size of 300 bp by 6 min sonication (40% power output, with pulses set to 30 sec ON / 10 sec OFF) in an ice-water bath using a Branson 250-D sonicator and a microtip. After sonication, Triton X-100 was added to 0.5% final concentration, and the lysate was centrifuged (5500 rpm, 5 min, 4°C) to remove cell debris. The chromatin extract was pre-cleared with 100 µl blocked (pre-absorbed with PBS/0.5% BSA) protein A/G sepharose beads for 2 h at 4°C, quantified in a UV spectrophotometer and diluted to 1 mg/ml and 0.25% N-lauryl sarcosine. 500 µl of the chromatin extract were used per single ChIP reaction in lubricated tubes in a total volume of 1000 µl. The extract was incubated overnight at 4°C with 50 µl blocked protein A/G sepharose beads which had been pre-adsorbed with 10 µg of antibody. Immune complexes were collected by centrifugation (3000 rpm, 1 min, 4°C) and washed six times with 1 ml of ice-cold ChIP wash buffer (50 mM Hepes-KOH pH 7.4, 500 mM LiCl, 1 mM EDTA, 1% NP40, 0.7% sodium deoxycholate, freshly added 0.5x protease inhibitor cocktail) and one time with 1x TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) containing 50 mM NaCl. The protein-DNA complexes were eluted from the beads by adding 200 µl ChIP elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) and incubation at 65°C under constant agitation for 10 min. After removal of beads by centrifugation (6000 rpm, 5 min, room temperature), the supernatant was incubated at 65°C overnight to revert the cross-links. Then, the sample was diluted to 400 µl with 1x TE. 8 µg of RNAse A were added and the sample was incubated for 1 hour at 37°C, followed by addition of proteinase K to 250 µg/ml and digestion for 2 hours at 55°C. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1% of the material used for ChIP) by phenol extraction and ethanol precipitation.
Label
Cy3
Label protocol
Probe labeling was performed by NimbleGen (Reykjavik, Iceland)
cell type: Epstein-Barr-Virus (EBV)-transformed human lymphoblastoid B cell line LCL 721 antibody: anti-TFIIB polyclonal rabbitt IgG antibody (sc-225)
Treatment protocol
standard growth conditions, no treatment
Growth protocol
LCL 721 cells were cultured for five passages (Px5) in RPMI 1640 medium at 37°C and 5% CO2 in a humidified incubator. Medium was supplemented with 10% heat-inactivated foetal bovine serum, 5 mM L-glutamine and 100 units/ml penicillin-streptomycin.
Extracted molecule
genomic DNA
Extraction protocol
1x10E8 cells (cell density 0.4x10E6 cells/ml) were pelleted by centrifugation (1200 rpm, 5 min), washed with PBS, and the pellet was resuspended in 36 ml of PBS (all steps at room temperature). Cells were fixed by adding 4 ml of a freshly prepared 10% formaldehyde solution (10% formaldehyde, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes-KOH pH 8.0). Cross-linking was done for 9 min at room temperature, followed by quenching with 125 mM glycine, with immediate transfer of the cell suspension to and 5 min incubation on ice. Cells were washed twice with ice-cold PBS and sequentially lysed by resuspending the cell pellet in 5 ml of ice-cold ChIP lysis buffer 1 (50 mM Hepes-KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100, freshly added 1x protease inhibitor cocktail) and 10 min rotation at 4°C. Cells were collected by centrifugation (4000 rpm, 10 min, 4°C), followed by resuspension in 5 ml of ice-cold ChIP lysis buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, freshly added 1x protease inhibitor cocktail) and 10 min rotation at 4°C. After centrifugation (4000 rpm, 10 min, 4°C), the pellet was resuspended in 3 ml of ice-cold ChIP lysis buffer 3 (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, freshly added 0.5% N-lauryl sarcosine, 0.1% sodium deoxycholate and 1x protease inhibitor cocktail). Acid-washed glass beads (212-300 microns) were added, and the crosslinked chromatin was sheared to an average size of 300 bp by 6 min sonication (40% power output, with pulses set to 30 sec ON / 10 sec OFF) in an ice-water bath using a Branson 250-D sonicator and a microtip. After sonication, Triton X-100 was added to 0.5% final concentration, and the lysate was centrifuged (5500 rpm, 5 min, 4°C) to remove cell debris. The chromatin extract was pre-cleared with 100 µl blocked (pre-absorbed with PBS/0.5% BSA) protein A/G sepharose beads for 2 h at 4°C, quantified in a UV spectrophotometer and diluted to 1 mg/ml and 0.25% N-lauryl sarcosine. 500 µl of the chromatin extract were used per single ChIP reaction in lubricated tubes in a total volume of 1000 µl. The extract was incubated overnight at 4°C with 50 µl blocked protein A/G sepharose beads which had been pre-adsorbed with 10 µg of antibody. Immune complexes were collected by centrifugation (3000 rpm, 1 min, 4°C) and washed six times with 1 ml of ice-cold ChIP wash buffer (50 mM Hepes-KOH pH 7.4, 500 mM LiCl, 1 mM EDTA, 1% NP40, 0.7% sodium deoxycholate, freshly added 0.5x protease inhibitor cocktail) and one time with 1x TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) containing 50 mM NaCl. The protein-DNA complexes were eluted from the beads by adding 200 µl ChIP elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) and incubation at 65°C under constant agitation for 10 min. After removal of beads by centrifugation (6000 rpm, 5 min, room temperature), the supernatant was incubated at 65°C overnight to revert the cross-links. Then, the sample was diluted to 400 µl with 1x TE. 8 µg of RNAse A were added and the sample was incubated for 1 hour at 37°C, followed by addition of proteinase K to 250 µg/ml and digestion for 2 hours at 55°C. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1% of the material used for ChIP) by phenol extraction and ethanol precipitation.
Label
Cy5
Label protocol
Probe labeling was performed by NimbleGen (Reykjavik, Iceland)
Hybridization protocol
Hybridization was performed by NimbleGen (Reykjavik, Iceland)
Scan protocol
Slides were scanned by NimbleScan (Reykjavik, Iceland)
Description
TFIIB_Px5
Data processing
Data were processed by NimbleGen according to standard procedures (see http://www.nimblegen.com). Each of the 24,134 human promoter regions is represented by a probe set consisting of 15x 50-mer individual oligonucleotide probes on the array. The median of the scaled log2 ChIP/input ratio of each probe set provides a measure for promoter occupancy. For each feature a log2 ratio of the hybridization intensities of the cohybridized ChIP-enriched sample (Cy5 channel) and non-enriched input sample (Cy3 channel) was determined. These ratios were scaled to center the data around zero by robust statistics. Specifically, scaling was performed by subtracting Tukey's bi-weight mean for the log2 ratios of all array features from each individual log2 ratio.
