Ribosol

Mice were euthanized with exposure to CO2 and sections of brain (suprachiasmatic nucleus (SCN) and immediately surrounding tissue), skeletal muscle, jejunum, colon, adipose tissue and lung were collected and flash frozen in liquid nitrogen. Total RNA was phenol extracted from an ~2mm diameter section of each tissue following mechanical homogenization in Ribosol (Amresco). RNA was then precipitated in isopropanol, washed twice with 75% and 95% EtOH, and resuspended in 200uL nuclease-free ddH20. An additional on-column DNase I treatment for 20 minutes at 23°C and RNAeasy spin-cup purification (Qiagen) following the manufacturer’s protocol was required to remove residual DNA carryover. RNA concentration was quantified with a Nanodrop spectrophotometer, and 1ug of total RNA was converted to cDNA at 42° for 30 minutes using a qScript cDNA synthesis kit as per manufacturer’s protocol (Quantabio).

RNA was extracted from S2 cells using either Ribosol (Amresco) or SIGMA RNA mini-prep per manufacturer’s protocol. Isolated RNA was DNase treated with Turbo DNase (Ambion) prior to cDNA synthesis. For cDNA synthesis 5x iScript Supermix (Bio-Rad) was used per manufacturer’s protocol. Quantitative PCR was performed with primers targeting Renilla luciferase or firefly luciferase, Supplemental Table 1. For 3′RACE: cDNA was synthesized using 3′RACE RT primer and 5x iScript Select (Bio-Rad) per manufacturer’s protocol. First round PCR was performed with Renilla-tail forward and 3′RACE External Amp primers. Second round PCR was performed with the overlap-forward primer for each 3′UTR being amplified (for example: GAPDH forward overlap) and 3′RACE amplification primer. The PCR products were resolved on an agarose gel. Prominent bands were excised and sequenced by Sanger sequencing. For qPCR of mature miRNAs we followed the protocol described previously^{65 (link)}. The primers used for this analysis are described in Table S3.
For analysis of p(A)-tail length we used the Poly(A) Tail-Length Assay Kit from Thermo-Fisher. The assay was performed per the manufacturer’s protocol. The primers used are described in Table S1: Hsp70a R, GAPDH R2 and HID/FLP F.