GSM9401869: Input of DRIPc-seq in RHKI, control with exogenous RNaseH treatment; Mus musculus; OTHER
RHKI cells were lysed in buffer (10 mM Tris, pH 7.5; 10 mM EDTA; 10 mM NaCl; 0.5% sarcosyl) with Proteinase K at 37°C overnight. DNA was extracted via PCI and ethanol precipitation, then digested with BsrGI, EcoRI, HindIII, SspI, and XbaI, followed by RNase III treatment for remove dsRNA (0.5U per 1µg gDNA). DNA was split into aliquots with or without RNase H (and RNase A if needed and 20-25U RNaseH per 1µg gDNA), re-extracted, and immunoprecipitated with 10-15 µg S9.6 antibody in MeDP buffer overnight at 4°C. Complexes were pulled down with Protein G beads, washed, and eluted in 2x STOP buffer (20 mM Tris 8.0, 100 mM NaCl, 20 mM EDTA, 1% SDS) at 65°C. DNA was purified and analyzed by qPCR using SYBR Fast reagent and specific primers. Enrichment was calculated as % input and fold change over negative control. Input DNA was used for library preparation using a NEBNext Ultra II kit with TruSeq adapters (no USER). Libraries were purified with double-size selection by Ampure XP beads (0.6x-0.9x). Immunoprecipitated RNA/DNA hybrids were digested with 10U DNase I and the RNA was recovered by PCI and ethanol precipitation. Libraries were prepared by NEBNext Ultra II Directional RNA library prep kit with TruSeq Adaptors, purified either with double-size selection by Ampure XP beads (0.6x-0.9x) or gel extraction (150-650 bp) and sequenced (NovaSeq, PE150; 90-100 million reads for DRIP libraries and 20-30 million reads for input libraries).