GSM5870408: KO_IP_RNaseR-_rep1; Mus musculus; OTHER
Totally 10^7 cells were trypsinized and centrifuged for the cell pellets. Cells were resuspended by 4 ml TE buffer with 0.5% SDS and 0.1 mg/ml proteinase K, incubated in constant temperature shaker at 37℃ for 16 h. Add 1/4 volume 5 M KAc, mix and place the tube on ice 10 min. Then the DNA was extracted by phenol: chloroform: isoamyl alcohol (25: 24: 1). Precipitate the supernatant by adding 1 volume isopropyl alcohol, 30 min at room temperature, and washed by 1 ml 70% ethanol. Dry the pellet and resuspend by 200 μl ddH2O. A cocktail of restrict enzymes (MseI, DdeI, AluI, MboI; NEB) was used for gDNA digestion in 200 μl system, incubated at 37°C overnight. Fragmented DNA was purified and quantified by Qubit 3.0 (Invitrogen). Mix 1.8 µg DNA with 0.2 µg E. coli DNA (for normalization) in 500 μl 1×TE. For RNase R treated group,  10 U RNase R (Epicentre) incubated at 37℃ for 1.5 h was used to remove linear RNA from DNA Fragment. For control group, the identical amount of reaction buffer was add to DNA Fragment incubated at 37℃ for 1.5 h. Take out 50 μl as Input, the rest 450 μl DNA was immunoprecipitated overnight at 4°C with 1× DRIP binding buffer (10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) and 10 µg S9.6 antibody (ATCC, HB-8730) at 500 μl system. Add 50 µl Protein G Dynabeads (Invitrogen, 10004D) and incubated for 4 h, then wash the beads by 1× DRIP binding buffer for 4 times at room temperature. Add 250 µl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS) and 5 U Proteinase K to the bead-antibody complexes, and incubated for 60 min in an Eppendorf ThermoMixer at 55°C. DNA from DRIP was purified and subjected to library construction using Accel-NGS® 1S Plus DNA Library Kit (Swift Biosciences). These libraries were utilized to 150 nt paired-end sequencing with an Illumina Nova 6000 PE150 system (Genewiz, China). Each library was generated from 70 to 121 million reads and then adapters were removed with cutadapt software to obtain cleandata.