title: GSM5260461: RbKO_100 J/m2 UVC_Replicate2; Homo sapiens; OTHER
library_construction_protocol: Wildtype and RB1 KO IMR-90 cells were grown to full confluency in order to eliminate cell cycle effects on UV lesion acquisition. CPD immunoprecipitation was performed as previously described (García‐Nieto et al, 2017). Cells were irradiated with 100J/m2 UVC and were immediately lysed with 1% SDS buffer. Cell lysates were treated with RNase A  and proteinase K , followed by DNA precipitation with ethanol/sodium acetate. Purified DNA was then sonicated by a Bioruptor (Diagenode), and heated to 99C to denature.  The resulting single-stranded fragments were incubated with anti-CPD antibody (Cosmos Bio, clone TDM-2). Immunoprecipitated DNA fragments were repaired by CPD and 6-4PP photolyases (Selby & Sancar, 2012). Libraries were prepped for Illumina sequencing using second‐strand DNA synthesis, end repair, and adapter ligation with NEBNext Directional Second‐Strand Synthesis (cat. no. E7550), and ChIP‐seq Library Prep Master Mix for Illumina (cat. no. E6240) kits