Experiment Overview
| Repository ID: | FR-FCM-Z5CQ | Experiment name: | Bay et al. 2022 Figure 5 | MIFlowCyt score: | 51.50% |
| Primary researcher: | Lilli Bay | PI/manager: | Helga Landsverk | Uploaded by: | Lilli Bay |
| Experiment dates: | 2022-05-01 - 2022-05-02 | Dataset uploaded: | May 2022 | Last updated: | Jun 2022 |
| Keywords: | [cell cycle] [EdU] [DRB] [RNAPII] [extracted cells] [ultraviolet radiation] [transcription] | Manuscripts: | [35641102] [35641102] [PMC9410883] | ||
| Organizations: |
Norwegian Radium Hospital, Oslo University Hospital, Radiation Biology, Oslo, --- Select One --- (Norway)
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| Purpose: | To address whether promoter proximal RNAPII is degraded on chromatin after UV by adding DRB prior to UV. | ||||
| Conclusion: | DRB prior to UV further enhanced removal of RNAPII from chromatin compared to cells treated with DRB or UV alone. The increase in chromatin levels of pRNAPII S5 was greater than the increase in pRNAPII S2 after treatment with UV + DRB + MG132 compared to UV + DRB, in line with more promoter proximal RNAPII being degraded than elongating RNAPII. DRB caused a relatively greater reduction in pRNAPII S2 levels in G1 and G2 phase compared to S phase after UV | ||||
| Comments: | Cells were treated with 1uM EdU for 30 min prior to inhibitor treatment + during treatment to allow cell cycle analysis. Cells were chromatin extracted prior to fixation to allow release of unbound factors. | ||||
| Funding: | This work was supported by the Norwegian Research Council (Grant #275918). | ||||
| Quality control: | Secondary antibody controls were included. Barcoded non-treated cells were included to minimize sample to sample variation during staining. In brief, non-treated HeLa cells were incubated with Alexa Fluor 647 Succinimidyl Ester. Barcoded cells were then distitributed equally among all the samples prior to antibody staining. All samples were normalized to the barcoded control upon analysis. CST run prior to analysis. | ||||
Experiment variables
| Conditions | |
|---|---|
| · Secondary antibody controls | export_HeLa_2nd Ab Rb_001_4.fcs · export_HeLa_2nd Ab Rb_001_5.fcs · export_HeLa_2nd Ab Rb_001_6.fcs · export_HeLa_2nd Ab rat_009_4.fcs · export_HeLa_2nd Ab rat_009_5.fcs · export_HeLa_2nd Ab rat_009_6.fcs |
| · non-treated | export_HeLa_RNAPII -_002_4.fcs · export_HeLa_RNAPII -_002_5.fcs · export_HeLa_RNAPII -_002_6.fcs · export_HeLa_pRNAPII S2 -_017_4.fcs · export_HeLa_pRNAPII S2 -_017_5.fcs · export_HeLa_pRNAPII S2 -_017_6.fcs · export_HeLa_pRNAPII S5 -_010_4.fcs · export_HeLa_pRNAPII S5 -_010_5.fcs · export_HeLa_pRNAPII S5 -_010_6.fcs |
| · UV 20 J/m2 2h | export_HeLa_RNAPII UV_003_4.fcs · export_HeLa_RNAPII UV_003_5.fcs · export_HeLa_RNAPII_UV_003_6.fcs · export_HeLa_pRNAPII S2 UV_018_4.fcs · export_HeLa_pRNAPII S2 UV_018_5.fcs · export_HeLa_pRNAPII S2 UV_018_6.fcs · export_HeLa_pRNAPII S5 UV_011_4.fcs · export_HeLa_pRNAPII S5 UV_011_5.fcs · export_HeLa_pRNAPII S5 UV_011_6.fcs |
| · DRB 2.5h | export_HeLa_RNAPII_DRB_007_4.fcs · export_HeLa_RNAPII_DRB_007_5.fcs · export_HeLa_RNAPII_DRB_007_6.fcs · export_HeLa_pRNAPII S2_DRB_022_4.fcs · export_HeLa_pRNAPII S2_DRB_022_5.fcs · export_HeLa_pRNAPII S2_DRB_022_6.fcs · export_HeLa_pRNAPII S5_DRB_015_4.fcs · export_HeLa_pRNAPII S5_DRB_015_5.fcs · export_HeLa_pRNAPII S5_DRB_015_6.fcs |
| · MG132 2.5h | export_HeLa_RNAPII_MG_008_4.fcs · export_HeLa_RNAPII_MG_008_5.fcs · export_HeLa_RNAPII_MG_008_6.fcs · export_HeLa_pRNAPII S2_MG_023_4.fcs · export_HeLa_pRNAPII S2_MG_023_5.fcs · export_HeLa_pRNAPII S2_MG_023_6.fcs · export_HeLa_pRNAPII S5_MG_016_4.fcs · export_HeLa_pRNAPII S5_MG_016_5.fcs · export_HeLa_pRNAPII S5_MG_016_6.fcs |
| · UV 20 J/m2 2h DRB and MG (added 30 min before UV) | export_HeLa_RNAPII_UV DRB and MG_006_4.fcs · export_HeLa_RNAPII_UV DRB and MG_006_5.fcs · export_HeLa_RNAPII_UV DRB and MG_006_6.fcs · export_HeLa_pRNAPII S2_UV DRB and MG_021_4.fcs · export_HeLa_pRNAPII S2_UV DRB and MG_021_5.fcs · export_HeLa_pRNAPII S2_UV DRB and MG_021_6.fcs · export_HeLa_pRNAPII S5 UV DRB and MG_014_4.fcs · export_HeLa_pRNAPII S5 UV DRB and MG_014_5.fcs · export_HeLa_pRNAPII S5 UV DRB and MG_014_6.fcs |
| · UV 20 J/m2 2h DRB (added 30 min before UV) | export_HeLa_RNAPII_UV and DRB_004_4.fcs · export_HeLa_RNAPII_UV and DRB_004_5.fcs · export_HeLa_RNAPII_UV and DRB_004_6.fcs · export_HeLa_pRNAPII S2 UV and DRB_019_4.fcs · export_HeLa_pRNAPII S2 UV and DRB_019_5.fcs · export_HeLa_pRNAPII S2 UV and DRB_019_6.fcs · export_HeLa_pRNAPII S5 UV and DRB_012_4.fcs · export_HeLa_pRNAPII S5 UV and DRB_012_5.fcs · export_HeLa_pRNAPII S5 UV and DRB_012_6.fcs |
| · UV 20 J/m2 2h MG132 (added 30 min before UV) | export_HeLa_RNAPII_UV and MG_005_4.fcs · export_HeLa_RNAPII_UV and MG_005_5.fcs · export_HeLa_RNAPII_UV and MG_005_6.fcs · export_HeLa_pRNAPII S2 UV and MG_020_4.fcs · export_HeLa_pRNAPII S2 UV and MG_020_5.fcs · export_HeLa_pRNAPII S2 UV and MG_020_6.fcs · export_HeLa_pRNAPII S5 UV and MG_013_4.fcs · export_HeLa_pRNAPII S5 UV and MG_013_5.fcs · export_HeLa_pRNAPII S5 UV and MG_013_6.fcs |
