title: Illumina HiSeq 1500 sequencing; RNA-seq of metabolically labelled RNA by 4-thiouracil from mouse tissues
design: RNA-seq of metabolically labelled RNA by 4-thiouracil from mouse tissues
library_construction_protocol: 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).