GSM4634561: adipocyte HFD mRNA rep 2; Mus musculus; RNA-Seq
RNA-seq: TRAP was performed as previously described (Roh et al., Cell Rep 2017). Adipose tissues were homogenized and processed to immunoprecipitation using anti-GFP antibody. Immunoprecipitates were washed and subjected to RNA extraction using Qiagen Micro RNeasy kit according to the manufacturer's instructions. Isolated RNA was quantified by Qubit, and RNA integrity was analyzed by Agilent Bioanalyzer. ChIP-seq: Tissues were dounce homogenized, and cross-linked nuclei were isolated and sorted by BD FACS Aria II using mCherry fluorescence. Sorted nuclei were sheared by Covaris E220 and used for ChIP for overnight. Immunoprecipitates were washed and subjected to elution and reverse crosslinking. DNA was then extracted by AMPure XP beads according to the manufacturer's manual. RNA-seq: TRAP-isolated RNA (≤100ng) was treated with the Ribo-Zero rRNA removal kit (Epicentre) to deplete ribosomal RNA and converted into double stranded cDNA using NEBNext mRNA Second Strand Synthesis Module. cDNA was subsequently tagmented and amplified for 12 cycles by using Nextera XT DNA Library Preparation Kit. Sequencing libraries were analyzed Qubit and Agilent Bioanalyzer, pooled at a final concentration of 12pM, and sequenced on a HiSeq2500. ChIP-seq: Sequencing libraries were constructed using the “on-bead” preparation method as previously described (Roh et al., Cell Rep, 2017). ChIP-isolated DNA was subjected to end repair/phosphorylation using the End-It DNA End-Repair Kit (Epicentre), A-tailing using the Klenow Fragment and index adaptor ligation using Quick Ligase (NEB). AMPure XP beads were left in all the reactions to allow isolation of DNA using PEG (Polyethylene Glycol 8000)/NaCl solution, thus minimizing loss between reactions. After ligation, DNA was eluted and amplified by PCR for 14-16 cycles using the PfuUltra II Hotstart PCR Master Mix. DNA sized between 250 and 600bp was selected by gel electrophoresis and extraction using E-Gel EX Agarose Gels (Invitrogen) and MinElute Gel Extraction kits (Qiagen). Libraries were analyzed using the Qubit and Agilent Bioanalyzer, pooled at a final concentration of 12pM and sequenced on a HiSeq2500 or NextSeq 500500 system.