GSM5857046: DRIP-seq; Mus musculus; OTHER
Genomic DNA for DRIP was isolated and digested as in Stork et al., 2016 and Ginno et al., 2012. RNase A treatment prior to DRIP was performed as in Stork et al., 2016. Digested DNA was phenol/chloroform-extracted, precipitated, washed and resuspended. For each DRIP reaction, 4.4 ug DNA were incubated with 10 ug S9.6 antibody for 16 h at 4 C, followed by incubation with magnetic protein G beads for 2 h at 4 C. Samples were washed for 3 x 10 min with 140 mM NaCl, 0.05% (w/v) Triton X-100, 10 mM NaPO4, pH 7.0, at room temperature and eluted in 10 mM EDTA, 0.5% (w/v) SDS, 50 mM Tris-Cl, pH 8.0 containing Proteinase K for 45 min at 55 C. DNA was phenol/chloroform-extracted and precipitated as described above. Per biological replicate, three repeat DRIP reactions were first analyzed separately by DRIP-qPCR to verify the DRIP procedure as in Sanz et al., 2019. Three DRIP reactions per biological replicate were then pooled for preparation of each DRIP-seq library. Input and DRIP DNA was sonicated to a size of ~300–700 bp and DRIP-seq library preparation was performed using NEB E6050 for end repair, NEB M0212 for A-tailing, and NEB E7335 for adapter ligation. 12 cycles of PCR were performed for library amplification and libraries were cleaned and size selected (Ampure XP beads).