GSM5914481: RNA PGC-1α-KD MB + FLAG-RNA PGC-1α(CBM5mut), rep #1; Mus musculus; RNA-Seq
Total RNA was extracted using TRIzolTM reagent, purified by ethanol precipitation Purified total cell-RNA was diluted in 2× Alkaline Fragmentation Solution (12 mM Na2CO3, 88 mM NaHCO3), incubated for 2−5-minutes at 95°C to generate ~20−100-nucleotide RNA fragments, 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).