GSM2342907: Mix sample_circRNA; Bos taurus; ncRNA-Seq
Total RNA was assessed by electrophoresis on a denaturing agarose gel and quantified by NanoDrop spectrophotometer (NanoDrop, USA) and Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). The total RNA samples (3 mg) were treated with the epicentre Ribo-ZeroTM Kit to remove rRNA before constructing the RNA-seq libraries. For RNase R treatment, ribosome-depleted RNA was incubated for 15 min at 37°C with 5 units μg−1 of RNase R (Epicentre Technologies, Madison, WI). Then the rRNA depleted RNA samples or circRNAs samples were fragmented and used to synthesize first- and second-strand complementary DNA (cDNA) with random hexamer primers, dNTPs and DNA Polymerase I. The cDNA fragments were cleaned and concentrated using AMPure XP beads, then the ends were repaired and modified with T4 DNA polymerase Klenow DNA polymerase to add A and adapter at the 3’ end of the DNA fragments. The ligated cDNA products were purified with AMPure XP beads and treated with uracil DNA glycosylase to remove the second-strand cDNA. Purified first-strand cDNA was subjected to PCR amplification, and the libraries were quality controlled with an Agilent 2100 Bioanalyzer and sequenced by HiSeq 2500 (Illumina, San Diego, CA) on a 150 bp paired-end (PE) run.
GEO Accession
GSM2342907