High Calorie_Post_M6
Bone marrow adipocytes were collected from bone marrow biopsy of the iliac crest, as previously described (Fazeli et al, JCI 2021). A subset of samples from the high calorie diet and fasting cohorts (High calorie diet pre n = 6, post n = 6; Fasting: pre n = 6, post n = 6) were shipped to the Vermont Biomedical Research Network (University of Vermont, Burlington, VT) for total RNA extraction and high-throughput RNA-sequencing analysis. RNA was extracted using a modified MagMAX mirVana RNA extraction protocol with a double clean up step added to remove excess phenol (Applied Biosystems A27828). Final RNA was protected by adding Ribolock RNase inhibitor (ThermoFisher, cat. no. EO0381, Waltham, MA USA) to a final concentration of 0.25U/L of RNA. RNA was evaluated for quality based on 28S and 18S rRNA using the RNA Picochip (Agilent cat. no. 50671513) on the Agilent Bioanalyzer 2100 (Santa Clara, Ca). The 260/280 ratio was assessed on a Nanodrop ND1000 (ThermoFisher, Waltham, MA) and quantified using the RNA HS kit using the Quantus Fluorometer (Promega Madison, WI). RNA-sequencing libraries for Illumina sequencing were constructed using 6 ng of total RNA with the Takara SMARTer Stranded Total RNA-Seq Kit v2 (634412) and evaluated using the Agilent Bioanalyzer 2100 and DNA HS chip. Equal amounts of each library were pooled for sequencing and combined with 10% PhiX control prior to sequencing using HiSeq 1500 with a single read V3 rapid flow cell for a total of 80bp (Illumina Corp San Diego, CA).
SUB15336347
M6_6_S6