GSM5912873: RIP N PGC-1α-KD MB + FLAG-RIP N PGC-1α(CBM5mut), rep #1; Mus musculus; RIP-Seq
Total RNA was extracted using TRIzolTM reagent, purified by ethanol precipitation "Lysates of cells were subjected to IP using anti-FLAG as previously described (Cho et al., 2018) with the following modifications. IPs utilized Anti-FLAG® M2 Magnetic Beads (Millipore), immunoprecipitates were washed twice with NET2 Buffer supplemented to 150 mM NaCl, twice with NET2 Buffer supplemented to 300 mM NaCl, and once with NET2 Buffer supplemented to 150 mM NaCl.  For native RIP-seq, contaminating genomic DNA was degraded using RQ1 RNase-free DNase in the presence of 0.4 U/µl of RNase OUT, and RNA was purified by phenol extraction and precipitated using ethanol. cDNA libraries were prepared using a TruSeq RNA Library Prep Kit v2 (Illumina) with the following modification: 20 ng of immunoprecipitated RNA was not subjected to poly(A)+ enrichment but rather directly fragmented, shortening the fragmentation time to 4 min.