GSM5866860: U2OS_siRAD51_MiDAS-seq; Homo sapiens; OTHER
After fixation, the cells were permeabilized with 0.2% Triton-X in PBS, and EdU was attached to a cleavable biotin linker (Azide-PEG(3 + 3)-S-S-biotin) (Jena Biosciences, Cat. No. CLK-A2112-10) using the reagents of the Click-it Kit (Invitrogen, Cat. No. C-10424). The DNA was isolated by phenol-chloroform extraction and ethanol precipitation and then sonicated to a size of 100–500 bp. Dynabeads MyOne streptavidin C1 (Invitrogen, Cat. No. 65001) were used to isolate the EdU-labeled DNA fragments. The beads were washed three times with Binding and Washing Buffer 1× (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.5% Tween-20) and then resuspended in twice their original volume with an equal volume of Binding and Washing Buffer 2× containing the sonicated DNA. The DNA-bead mixture was incubated 15 min at room temperature with rotation, prior to washing the beads three times with Binding and Washing Buffer 1× and once with TE buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). To elute the EdU-labeled DNA from the streptavidin beads, 2% β-mercaptoethanol (Sigma, Cat. No. M6250) was added for 1 h at room temperature. The eluted DNA was finally used for library preparation. Sequencing libraries were made by the Genomics Platform of the University of Geneva using the TruSeq ChIP Sample Prep Kit (Illumina, Cat. No. IP-202-1012). Please note that previously published (PMID: 32561860) MiDAS-seq samples from U2OS cells treated with low and high doses of Aphidicolin were collected concurrently with the siRAD51 sample preparation and are accessible from the Sequence Read Archive as BioProject PRJNA588267.