Journal: bioRxiv

Article Title: Coordination of transcription-coupled repair and repair-independent release of stalled RNA polymerase II in response to transcription-blocking lesions

doi: 10.1101/2024.07.07.602436

Figure Lengend Snippet: a, Schematic representation of Damage-seq and PADD-seq. For Damage-seq, genomic DNA extracted from UV-irradiated cells are sonicated and ligated to the first adaptor, followed by immunoprecipitation with the lesion-specific antibody. The precise sites of the lesions are determined by the stalling of primer extension with high-fidelity DNA polymerase. After the ligation of the second adaptor, the primer extension products are PCR-amplified and sequenced. For PADD-seq, PolII-associated DNA are enriched by regular chromatin- immunoprecipitation (ChIP) with an anti-pan-RPB1 antibody. Purified DNA fragments are subjected to Damage-seq. b, Diagram of the experimental design for the measurement of TCR (by Damage-seq) and PolII-CPD interaction (by PADD-seq). c, Meta-gene analysis of PADD-seq signals around TSSs and TESs for active genes longer than 50 kb ( n = 2790) under indicated conditions. d, Quantification of PolII retention on damage sites by relative change of PADD-seq signals from 0.5 h to 2 h on each gene. Active genes longer than 20 kb were selected ( n = 4488). e, Meta-gene analysis of Damage-seq signals around TSSs for active genes longer than 50 kb ( n = 2790) under indicated conditions. Cells were collected immediately (0 h) or at 8 h after UV irradiation. Fraction CPDs remaining was calculated as the ratio of 8 h to 0 h. f, Violin plots of relative Damage-seq signals on each active gene ( n = 6406). Log2 value of the ratio of fraction CPDs remaining on TS to that on NTS was calculated. P value was calculated using two-tailed paired Student’s t -test for (d) and (f) . See also Supplementary Fig. 1 and 2. Source data are provided as a Source Data file.

Article Snippet: In brief, 167 μg of fragmented chromatin and 25 μg of anti-RPB1 antibody (Bethyl Laboratories, A304-405A) were incubated in RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% SDS and 0.1% Na-DOC) supplemented with protease inhibitors, 0.1 % BSA (Sigma) and 0.1 μg/μl tRNA (Sigma) for 2 h on a rotator at 4 °C.

Techniques: Irradiation, Sonication, Immunoprecipitation, Ligation, Amplification, Chromatin Immunoprecipitation, Purification, Two Tailed Test

Journal: bioRxiv

Article Title: Coordination of transcription-coupled repair and repair-independent release of stalled RNA polymerase II in response to transcription-blocking lesions

doi: 10.1101/2024.07.07.602436

Figure Lengend Snippet: a, Meta-gene analysis of Damage-seq signals around TSSs for active genes longer than 50 kb ( n = 2790) under indicated conditions. Cells were collected immediately (0 h) or at 8 h after UV irradiation. Fraction CPDs remaining was calculated as the ratio of 8 h to 0 h. Data of WT and CSB -KO cells are from our previous study . b, Violin plots of relative Damage-seq signals on each active gene ( n = 6406). Log2 value of the ratio of fraction CPDs remaining on TS to that on NTS was calculated. c, Meta-gene analysis of PADD-seq signals around TSSs and TESs for active genes longer than 50 kb ( n = 2790) under indicated conditions. d, Quantification of PolII retention on damage sites by relative change of PADD-seq signals from 0.5 h to 2 h on each gene. Active genes longer than 20 kb were selected ( n = 4488). P value was calculated using two-tailed paired Student’s t -test for (b) and (d) . e, Diagram showing that three possible fates of lesion-stalled PolII should co-exist in RPB1-K1268R cells. See also Supplementary Fig. 5. Source data are provided as a Source Data file.

Article Snippet: In brief, 167 μg of fragmented chromatin and 25 μg of anti-RPB1 antibody (Bethyl Laboratories, A304-405A) were incubated in RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% SDS and 0.1% Na-DOC) supplemented with protease inhibitors, 0.1 % BSA (Sigma) and 0.1 μg/μl tRNA (Sigma) for 2 h on a rotator at 4 °C.

Techniques: Irradiation, Two Tailed Test