title: GSM8714967: mouse degradome seq Heart WT 1646; Mus musculus; OTHER
library_construction_protocol: Tissues were extracted and frozen on dry ice. A bead mill (Retsch, Düsseldorf, Germany) was used to pulverize the tissue. RNA was subsequently extracted from the tissue powder using a phenol-based extraction protocol (PeqGold TRIFAST, VWR) according to the manufacturer's protocol. RNA integrity was visualized using a Bioalalyzer (Agilent, Santa Clara, CA, USA) The protocol was adapted from Li et al (doi.org/10.1186/s13007-019-0524-7) but using oligo nucleotides compatible with NEBNext® Multiplex Oligos for Illumina (NEB, Ipswich, MA, USA). Poly(A) RNA was isolated from 5 µg total RNA using magnetic beads (NEBNext® Poly(A) mRNA Magnetic Isolation Module, NEB, Ipschwich, MA, USA). Uncapped RNA was then ligated to an adapter and reverse transcribed using a poly(A) binding primer. After second strand synthesis and purification, the double stranded fragments were digested with EcoP15I and purified before a double stranded adapter was ligated to the fragment containing the EcoP15I recognition site. After purification and amplification these fragments were prepared for standard Illumina single-read sequencing using a small RNA sequencing protocol.