title: GSM4969219: C2C12P_Cytosol_Polysome_rep1; Mus musculus; OTHER
library_construction_protocol: Phenol/Choloroform RNAseq libraries: 250 ng of total RNA was used for library construction using NEBNext® Ultra™ II RNA Library Prep Kit for Illumina®. 3'READS+ libraries: The READS+ procedure has been previously described (Zheng et al., 2016). Briefly, polyA+ RNA in 0.1 ~ 2.5 µg of total-cell RNA was captured using 10 µl of oligo(dT)25 magnetic beads (NEB) in 100 µl of 1x Binding Buffer (10 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM EDTA, and 0.05% TWEEN 20) and fragmented on the beads using 1.5 U of RNase III (NEB) in 30 µl of RNase III Buffer (10 mM Tris-Cl pH 8.3, 60 mM NaCl, 10 mM MgCl2, and 1 mM DTT) for 15 min at 37 °C. After washing away unbound RNA fragments using Binding Buffer, polyA+ fragments were eluted from the beads using TE Buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.5) and precipitated using ethanol, followed by ligation to 3 pmol of heat-denatured 5'-adapter (5'-CCUUGGCACCCGAGAAUUCCANNNN-3') in the presence of 1 mM ATP, 0.1 µl of SuperaseIn (ThermoFisher Scientific), and 0.25 µl of T4 RNA ligase 1 in a 5 µl reaction at 22 °C for 1 hr. Ligation products were captured using 10 pmol of biotin-T15-(+TT)5 attached to 12 µl of Dynabeads MyOne Streptavidin C1 (ThermoFisher Scientific). After washing with Washing Buffer (10 mM Tris-Cl pH 7.5, 1 mM NaCl, 1 mM EDTA, and 0.05% TWEEN 20), RNA fragments on the beads were incubated with 0.01 U/µl of RNase H (Epicentre) for 30 min at 37 °C in 30 µl of RNase H buffer (50 mM Tris-Cl pH 7.5, 5 mM NaCl, 10 mM MgCl2, and 10 mM DTT). After washing with RNase H Buffer, RNA fragments were eluted from the beads in Elution Buffer (1 mM NaCl, 1 mM EDTA pH7.5, and 0.05% TWEEN 20) at 50 °C, precipitated using ethanol, and then ligated to 3 pmol of heat-denatured 5'-adenylated 3'-adapter (5'-rApp/NNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC-3') using 0.25 µl of T4 RNA ligase 2 (truncated KQ version) for 1 hr at 22 °C in a 5 µl reaction containing 15% PEG 8000 and 0.2 µl of SuperaseIn. Ligation products were precipitated and reverse-transcribed using M-MLV reverse transcriptase, and PCR-amplified using Phusion high-fidelity DNA polymerase (NEB) and bar-coded PCR primers (Sigma-Aldrich) for 15 cycles. Both RT primer (5'-GTTCAGAGTTCTACAGTCCGACGATC-3') and PCR primers with indexes have been described previously (Hoque et al., 2013). PCR products were size-selected twice using 0.6 volume of AMPure XP beads (Beckman Coulter) relative to the PCR reaction volume to remove large DNA molecules, followed by an additional 0.4 volume of beads to remove small DNA molecules. The eluted DNA was selected again with 1 volume of AMPure XP beads to further remove small DNA molecules. The sizes of the libraries eluted from the AMPure beads were examined using a high-sensitivity DNA kit (Agilent) and an Agilent Bioanalyzer. Library concentrations were measured using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific). Equal amounts of libraries were mixed and sequenced using an Illumina NextSeq 500 (1x75 bases).