Illumina HiSeq 1500 sequencing; RNA-seq of metabolically labelled RNA by 4-thiouracil from mouse tissues
RNA-seq of metabolically labelled RNA by 4-thiouracil from mouse tissues
The mice were culled by terminal anaesthesia with isoflurane followed by cardiac puncture. The death was confirmed by cervical dislocation. Tissues were cut into small pieces (less than 5 mm in thickness) and submerged in RNAlater (Sigma-Aldrich) for storage at -20C. After removing RNAlater by clean Kimtech (Kimberly-Clark), around 30 mg of tissue was homogenised in 1 ml TRIsure (Bioline), using TissueLyser LT (Qiagen) and 7 mm Stainless Steel Beads (Qiagen). RNA extraction was performed following the manufacturer's instruction with minor modifications: the isopropanol precipitation step was done with the addition of 20 ug/ml glycogen and 0.1 mM DTT and the isopropanol precipitation at -20C was extended to 2 hr. Purified RNA was subsequently treated with 1 U of TURBO DNase (Invitrogen) to eliminate potential DNA contamination. After the DNase reaction, RNA was cleaned up by RNA Clean & Concentrator-5 (Zymo Research) and stored at -80C with 1 mM DTT in order to prevent oxidisation.  DNase-treated RNA was reacted with IAA to alkylate the thiol group following the protocol previously described. Briefly, 50 µl reaction mix (5-10 µg RNA, 10 mM IAA, 50 mM pH8 sodium phosphate, and 50% DMSO) was incubated at 50C for 15 min. The reaction was stopped by adding 1 µl of 1M DTT, followed by 1 µl glycogen (20 mg/ml), 5 µl NaOAc (3M, pH 5.2), and 125 µl 100% EtOH addition. After 2 hr incubation at -20C, the solution was centrifuged and the obtained RNA pellet was washed with 80% ethanol. The pellet was resuspended in 15 ul nuclease-free water. RNA concentration was quantified by Qubit RNA BR Assay Kit (Molecular probes) and 500 ng of it was used as an input for QuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina (Lexogen). RNA-seq library preparation was conducted following manufacturer's instructions with the PCR cycles optimised for different tissues (brain: 13, duodenum: 15, eWAT: 15).
Experimental Factor: genotype
uprt/0; +/+
Experimental Factor: organism part
brain