Five micrograms of total RNA was pretreated with either 20 U of TAP (Epicentre) or 20 U of RNA 5′ polyphosphatase (Epicentre) in a volume of 20 µL for 45 min at 37°C or used directly. After standard extraction, RNA was used for TruSeq small RNA library preparation according to the manufacturer's instructions (Illumina), including 15 cycles of PCR. Libraries were sequenced on a MiSeq instrument (Illumina). A detailed description of processing and analysis of the data thus generated are in the Supplemental Material.
Purification of nuclear short RNAs was performed as described (Chen et al. 2013 (link)). Briefly, nuclei of 2 million young adults grown in liquid culture were isolated as described in Ooi et al. (2010) (link), and RNA was extracted using Tripure (Roche). Short capRNA-seq libraries were cloned from 20 µg of nuclear RNA as follows: After size selection for RNAs of 20–100 nt, we performed RNA polyphosphatase (Epibio) treatment followed by Terminator exonuclease (Epibio) treatment and 3′ adapter ligation. After treatment with heat labile alkaline phosphatase (Epibio), capped RNAs were rendered accessible for cloning by TAP treatment. 5′-adapter cloning and library generation were completed as described in the TruSeq small RNA kit (Illumina), and sequencing was performed on a HiSeq instrument (Illumina, SE50).
Purification of nuclear short RNAs was performed as described (Chen et al. 2013 (link)). Briefly, nuclei of 2 million young adults grown in liquid culture were isolated as described in Ooi et al. (2010) (link), and RNA was extracted using Tripure (Roche). Short capRNA-seq libraries were cloned from 20 µg of nuclear RNA as follows: After size selection for RNAs of 20–100 nt, we performed RNA polyphosphatase (Epibio) treatment followed by Terminator exonuclease (Epibio) treatment and 3′ adapter ligation. After treatment with heat labile alkaline phosphatase (Epibio), capped RNAs were rendered accessible for cloning by TAP treatment. 5′-adapter cloning and library generation were completed as described in the TruSeq small RNA kit (Illumina), and sequencing was performed on a HiSeq instrument (Illumina, SE50).
Free full text: Click here
