GSM6569762: WT HFD adipocytes mRNA rep. 3; Mus musculus; RNA-Seq
TRAP was conducted as previously described (Roh et al., 2017). Pieces of frozen epididymal adipose tissue (100mg) were homogenized by dounce (7ml) in homogenization buffer (50mM Tris [pH7.5], 12mM MgCl2, 100mM KCl, 1% NP-40; 100mg/ml cycloheximide, 1mg/ml sodium heparin, 2mM DTT, 0.2units/ml RNasin, 1x Roche Complete EDTA-free protease inhibitor), and incubated on ice for 15 min with vortexing every 5 min. Lysates were centrifuged at 13,000rpm for 10min and the supernatant was collected after removing the top lipid layer. Anti-GFP antibody (5mg/ml, Abcam ab290) was added to the collected supernatant and rotated for 1h at 4°C. Protein G dynabeads washed twice with low-salt wash buffer (50mM Tris [pH7.5], 12mM MgCl2, 100mM KCl, 1% NP-40, 100mg/ml cycloheximide, 2mM DTT) were added to the lysates with antibody and further rotated for 1h at 4°C. The immunoprecipitates were washed three times in high-salt wash buffer (50mM Tris [pH 7.5], 12mM MgCl2, 300mM KCl, 1% NP-40, 100mg/ml cycloheximide, 2mM DTT) on a magnet. After the ﬁnal wash, immunoprecipitates were subjected to RNA extraction using the Qiagen Micro RNeasy kit according to the manufacturer's instructions. For RNA-seq library construction, TRAP-isolated RNA (≤100ng) and SVF-derived adipocyte RNA (100ng) were treated with the Ribo-Zero rRNA removal kit (Epicentre, USA) to deplete ribosomal RNA and converted into double-stranded cDNA using NEBNext mRNA Second Strand Synthesis Module (E6111L). cDNA was subsequently segmented and ampliﬁed for 12 cycles by using Nextera XT DNA Library Preparation Kit (Illumina FC-131, USA). Sequencing libraries were analyzed by Qubit and Agilent Bioanalyzer, pooled at a ﬁnal concentration of noon, and sequenced on a HiSeq2500.