title: GSM5912313: RIP FP PGC-1α-KD MB + FLAG, rep #2; Mus musculus; RIP-Seq
library_construction_protocol: 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 RIP-seq footprinting, washed immunoprecipitates were digested on-bead using 2 U/μL RNase I for 30 minutes at 4°C so as to generate ~20−100-nucleotide RNP fragments, i.e. PGC-1α or control RNA footprints. Immunoprecipitated RNA fragments were purified using TRIzol and precipitated using ethanol. Contaminating genomic DNA was degraded using RQ1 RNase-free DNase (Promega) in the presence of 0.4 U/µl of RNase OUT (Invitrogen). RNA was purified by phenol extraction and precipitated using ethanol. DNA-free RNA (500 ng) was decapped using RNA 5´ pyrophosphohydrolase (RppH; New England Biolabs), purified using TRIzol extraction, and precipitated overnight at 4°C using isopropanol. RNA 5´ and 3´ ends were dephosphorylated using recombinant Shrimp Alkaline Phosphatase (rSAP) (New England Biolabs), rSAP was heat-inactivated, and RNA 5´ends were re-phosphorylated using T4 polynucleotide kinase (T4 PNK, New England Biolabs) in the presence of RNase OUT. RNA with repaired ends was purified using Monarch® RNA Cleanup Kit (New England Biolabs). cDNA libraries were prepared using a NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs, Version 8.0_10/20 protocol). PCR products of 140–300-bp were size-selected using Pippin HT (Sage Science).