strain: delta_flap_Rpb2 cell line: HEK293_Tet_ON_Clonetech chip antibody: FLAG
Treatment protocol
Delta-Flap Rpb2-FLAG cells were treated with doxycycline (2 μg/mL).
Growth protocol
Delta-Flap Rpb2-FLAG cells were grown as monolayers in DMEM supplemented with Tet free FBS and sodium bicarbonate (0.375%) and hygromycin (200 μg/mL).
Extracted molecule
genomic DNA
Extraction protocol
ChIP assays were performed essentially as described elsewhere. Cells were grown on 10 cm2 dishes, crosslinked by addition of formaldehyde to 1% and incubation at room temperature for 15 min, washed twice with PBS, scraped off the plates, and centrifuged in 15 ml conical tubes for 10 min at 1000 g. The pelleted cells were transferred to a 1.5 ml microfuge tube in 550 µl buffer A (50 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl and 1% Triton X-100) and sonicated (microtip sonicator, 60% output) 8 times on ice, pulsed for 30 s. Cell debris was removed by centrifugation at 20,800 g for 10 min at 4 oC, and the sample was then pre-cleared by addition of 30 µl Pansorbin (fixed Protein A-bearing S. aureus cells; Calbiochem) and incubation continued at 4 oC for 1 hour. RNAPII was immunoprecipitated with M2 anti-Flag antibody or with 8WG16 monoclonal antibody (45). Antibody (2-5 µg) was added to the pre-cleared sample and incubated overnight at 4 °C with mixing. Antibody complexes were recovered by binding to 30 µl Pansorbin and centrifugation for 4 min at 20,800 g, and then washed sequentially with buffer A, buffer B (50 mM Tris-HCl, pH 7.5, 750 mM KCl, 1 mM EDTA, 10% glycerol and 1% Triton X-100), and buffer C (20 mM Tris-HCl, pH 8.0, 100 mM KCl, 10 mM β−mercaptoethanol, 0.2 mM EDTA, 20% glycerol and 0.1% Triton X-100). Captured complexes were released by incubation for 20 min at room temperature in elution buffer (10% SDS, 100 mM NaHCO3), and recovery of supernatant after centrifugation at room temperature for 10 min at 20,800 g. The Pansorbin pellet was washed an additional time and the two washes were combined. Crosslinks were reversed by incubating at 65 °C overnight and the released DNA was then purified using a QIAquick PCR purification column.
Label
Cy5
Label protocol
The immunoprecipitated (IP) DNA sample and an input DNA sample (recovered after similar treatment but without immunoprecipitation) were amplified by linker-mediated PCR as described in the protocol from Nimblegen. The amplified DNA was then labeled with Cy3 (input DNA) or Cy5 (IP DNA) dye.
strain: delta_fl_Rpb2 cell line: HEK293_Tet_ON_Clonetech chip antibody: none, input DNA
Treatment protocol
Delta-Flap Rpb2-FLAG cells were treated with doxycycline (2 μg/mL).
Growth protocol
Delta-Flap Rpb2-FLAG cells were grown as monolayers in DMEM supplemented with Tet free FBS and sodium bicarbonate (0.375%) and hygromycin (200 μg/mL).
Extracted molecule
genomic DNA
Extraction protocol
ChIP assays were performed essentially as described elsewhere. Cells were grown on 10 cm2 dishes, crosslinked by addition of formaldehyde to 1% and incubation at room temperature for 15 min, washed twice with PBS, scraped off the plates, and centrifuged in 15 ml conical tubes for 10 min at 1000 g. The pelleted cells were transferred to a 1.5 ml microfuge tube in 550 µl buffer A (50 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl and 1% Triton X-100) and sonicated (microtip sonicator, 60% output) 8 times on ice, pulsed for 30 s. Cell debris was removed by centrifugation at 20,800 g for 10 min at 4 oC, and the sample was then pre-cleared by addition of 30 µl Pansorbin (fixed Protein A-bearing S. aureus cells; Calbiochem) and incubation continued at 4 oC for 1 hour. RNAPII was immunoprecipitated with M2 anti-Flag antibody or with 8WG16 monoclonal antibody (45). Antibody (2-5 µg) was added to the pre-cleared sample and incubated overnight at 4 °C with mixing. Antibody complexes were recovered by binding to 30 µl Pansorbin and centrifugation for 4 min at 20,800 g, and then washed sequentially with buffer A, buffer B (50 mM Tris-HCl, pH 7.5, 750 mM KCl, 1 mM EDTA, 10% glycerol and 1% Triton X-100), and buffer C (20 mM Tris-HCl, pH 8.0, 100 mM KCl, 10 mM β−mercaptoethanol, 0.2 mM EDTA, 20% glycerol and 0.1% Triton X-100). Captured complexes were released by incubation for 20 min at room temperature in elution buffer (10% SDS, 100 mM NaHCO3), and recovery of supernatant after centrifugation at room temperature for 10 min at 20,800 g. The Pansorbin pellet was washed an additional time and the two washes were combined. Crosslinks were reversed by incubating at 65 °C overnight and the released DNA was then purified using a QIAquick PCR purification column.
Label
Cy3
Label protocol
The immunoprecipitated (IP) DNA sample and an input DNA sample (recovered after similar treatment but without immunoprecipitation) were amplified by linker-mediated PCR as described in the protocol from Nimblegen. The amplified DNA was then labeled with Cy3 (input DNA) or Cy5 (IP DNA) dye.
Hybridization protocol
The amplified DNA was then labeled with Cy3 (input DNA) or Cy5 (IP DNA) dye, hybridized to a Nimblegen High Density 2.1 M promoter tiling array using a Maui hybridization apparatus (BioMicro Systems), and quantified using a Axon 4000B scanner (Molecular Devices) following the protocol from Nimblegen.
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.3 ChIP data extraction. Eight ChIP datasets were generated as log2(IP/input) fluorescence intensities using anti-FLAG IP of wild-type RPB2-FLAG cells in biological triplicate, anti-FLAG IP of ∆FL RPB2 FLAG cells in biological triplicate, and anti-RPB1 CTD, 8WG16 of both wild-type and ∆FL cells in biological duplicate. The two 8WG16- IP datasets were combined to measure the distribution of RPB1 signal. Data analysis was performed using the statistical program R (Bioconductor) and publicly available packages. To compare RNAPII distributions in wild-type and ∆FL RPB2 cells, replicate data sets were quantile normalized using the normalizeQuantiles function in the affyPLM package, averaged at each probe position to generate single values for each target, and then quantile normalized against each other to allow comparison.
