GSM7899196: RNA-seq, Beige Ad. Diff. 0d, rep2; Mus musculus; RNA-Seq
Cultured cells were washed two times with 1X Phosphate-base buffer (PBS) and then incubated for 30 min on ice with the lysis buffer (10 mM/mL Tris-HCl, pH 7.4 (AM9850G, AM9855G, Invitrogen); 5 mM/mL MgCl2 (#AM9530G, Invitrogen); 100 mM/mL KCl (#AM9640G, Invitrogen); 2 mM/mL dithiothreitol (DTT, #707265ML, Thermo Fisher); 100 μg/mL cycloheximide (CHX, #C1988, Sigma‒Aldrich); 1% Triton X-100 (#T8787, Sigma‒Aldrich); 1X protease inhibitor (#539134, Sigma-Aldrich); 2 μL/mL SUPERase inhibitor (#AM2696, Invitrogen); and 2 μL/mL RNase inhibitor (#AM2694, Invitrogen). After centrifuging the lysates at 20000 g for 15 min at 4 °C, the supernatant was separated into three portions for RNA-seq, Ribo-Seq, and Mass Spectrometry. For RNA-seq, the supernatant was treated with TRIzol LS (#10296028, Invitrogen) to isolate RNA. After the purification of total RNA, libraries were constructed with TruSeq Stranded Total RNA Library Prep Gold (#20020599 Illumina) as per the manufacturer's protocol, followed with sequencing via NovaSeq 6000 system.