GSM7014975: TH_m181; Mus musculus; RNA-Seq
Whole-tissue RNA extraction was performed as described previously (Surya et al., 2020). Briefly, tissues stored in RNA-preserving solution were thawed and transferred to 2 mL tubes containing 700-1500 µL (depending on tissue) of PureZOL (Bio-Rad, 7326890) or homemade Trizol-like solution (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Tissues were lysed by adding 2.8-mm ceramic beads (OMNI International, 19-646) and running 1-3 cycles of 5-45 s at 3500 rpm on the PowerLyzer 24 (QIAGEN). For liver, brain, and small intestine samples, tissues were lysed with 3-5 mL using M tubes (Miltenyi biotec, 130-096-335) and running 1-4 cycles of the RNA_02.01 program on the gentleMACS Octo Dissociator (Miltenyi biotec). Next, lysates were processed in deep 96-well plates (USA Scientific 1896-2000) by adding chloroform for phase separation by centrifugation, followed by precipitation of total RNA in the aqueous phase using magnetic beads coated with silane (Dynabeads MyOne Silane; TermoFisher Scientific 37002D), buffer RLT (QIAGEN 79216), and ethanol. Genomic DNA contamination was removed by on-bead DNase I (ThermoFisher Scientific AM2239) treatment at 37 °C for 20 min. After washing steps with 80% ethanol, RNA was eluted from beads. This RNA extraction protocol was performed on the Bravo Automated Liquid Handling Platform (Agilent). Sample concentrations were measured using a Nanodrop One (Termo Scientific). RNA quality was confirmed using a Tapestation 4200 (Agilent Technologies). For each tissue sample, full-length cDNA was synthesized in 20 µl final reaction volume containing the following: (1) 10 µl of 10 ng/µl RNA; (2) 1 µl containing 2 pmoles of a custom RT primer biotinylated in 5'