title: GSM9320990: DRIPc_RNaseH_siNTC_RBRIIImut; Mus musculus; OTHER
library_construction_protocol: DRIPc-sequencing was adapted from Sanz and Chédin, 2019. Cells were lysed rotating overnight at 37°C in TE buffer (pH 8) supplemented with 10% SDS and 10 µL proteinase K (10 mg/mL). For spike-normalization 10% Drosophila S2 cells were added according to the cell number. DNA was isolated by phenol/chloroform extraction using MaXtract High Density Tubes (Qiagen) and fragmented with a mixture of restriction enzymes. For this DNA was incubated overnight with 60 U of BsrGI, EcoRI, HindIII, SSpI and Xbal (New England Biolabs) at 37°C. DNA was cleaned up again with phenol/chloroform and fragmentation was validated by agarose gel electrophoresis to ensure successful digestion of the DNA to a size below 1 kb. Subsequently, 200 µg of DNA was treated with 30 U of RNaseT1 (Thermo Fisher Scientific) at 37°C for 30 min to eliminate ssRNA. To avoid potential dsRNA contamination the DNA was treated with 3 U RNaseIII (Thermo Fisher Scientific) for 1 h at 37°C. Samples were then incubated for 6 h at 37°C with or without 60 U of RNaseH1 (New England Biolabs) to ensure specificity of the following immunoprecipitation step. For this DNA in 1 x DRIP binding buffer (10 x DRIP binding buffer: 100 mM Sodium phosphate, 1.4 M NaCl, 0.5 % Triton X-100) was split into two tubes and incubated overnight with 4 µL of S9.6 antibody (New England Biolabs) while rotating. For total input samples, 10 % of DNA was taken before the immunoprecipitation. On the next day wash 40 µL/IP of Protein A and Protein G-coupled Dynabeads (Thermo Fisher Scientific), add to the DNA/antibody mix and incubate for 4 h on a rotating wheel at. 4°C Immunoprecipitated complexes were then washed 3 times each with ice-cold 1 x DRIP binding buffer. Finally, DNA was eluted by adding DRIP elution buffer (50 mM TRIS pH 8,10 mM EDTA, 0.5 % SDS) and roteinase K for 45 min at 55°C while shaking at 900 rpm. DNA was purified with phenol/chloroform extraction and finally eluted in 50 µl TE buffer. Before proceeding to library preparation, 4 µL were used to test enrichment by qPCR. DNA:RNA hybrids were then treated with DNase (Thermo Fisher Scientific) for 45 min at 37°C. DNase was inactivated by inactivation buffer (Thermo Fisher Scientific) for 5 min at room temperature. After 5 min of centrifugation at 10'000 rpm the supernatant was transferred to a new PCR tube and libraries were generated using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) following manufacturer's instructions.