Dynabeads oligo t magnetic beads

In the present study, we used our WTTS-Seq protocol^{23 (link),81 (link)} to construct libraries for all rats. For each individual sample, 2.5 μg of total RNA was chemically fragmented using RNA fragmentation buffer (AM8740, Ambion) as the first step, then enrichment of poly(A) + RNA was accomplished with Dynabeads oligo(T) magnetic beads (61002, Ambion) followed by reverse transcription with SuperScript III Reverse Transcriptase (18080, Invitrogen) to synthesize first strand cDNA. Next, both 5′- and 3′- adaptors were added to fit the Ion Torrent sequencing platform. All RNA molecules were removed by both RNases H (M0297L, NEB) and I (EN0601, Thermo Scientific) to prevent contamination, 250–500 bp first-strand cDNA molecules were selected with solid-phase reversible immobilization beads (A63880, Beckman Coulter), and second-strand cDNA was synthesized by PCR. Lastly, all libraries were sequenced using an Ion PGM™ Sequencer at Washington State University.

In the present study, two male KO and two male WT mice were used per gene model. For each individual, 2.5 μg of total RNA derived from muscle were chemically fragmented with RNA fragmentation buffer (AM8740, Ambion). After that, poly(A)+ RNA was enriched by Dynabeads oligo(T) magnetic beads (61002, Ambion). Reverse transcription with SuperScript III Reverse Transcriptase (18080, Invitrogen) was used to synthesize first cDNA strand with integration of both 5′adaptor and 3′adaptor. After all RNA molecules were removed by both RNases H (M0297L, NEB) and I (EN0601, Thermo Scientific), solid-phase reversible immobilization beads were used to select 300–600 bp first-strand cDNA molecules (A63880, Beckman Coulter), and second-strand cDNA was synthesized by PCR. Size selection was repeated as described above and the final libraries were sequenced using an Ion PGM™ system at Washington State University.