GSM2771219: 04_N22_TAP; Caenorhabditis elegans; miRNA-Seq
Animals were collected and subsequently digested with Proteinase K. Total RNA was acquired using Trizol LS reagent, according to the manufacturer's instructions. Samples were enriched for sRNAs using a mirVana Kit (Life Technologies #AM1561). Each sRNA enriched sample was treated in three different ways: 1) directly cloned, 2) treated with Tobacco Acid Phosphatase (TAP), and 3) Oxidized. 1 µg of RNA was treated with 5 U of tobacco acid phosphatase (Epicenter) at 37°C for 2 h to digest 5’ tri- and di-phosphates to mono-phosphates. To oxidate sRNAs, 3 µg of sRNA enriched sample was incubated for 10 minutes at room temperature with 200 mM NaIO4, 5X borate buffer (148 mM Borax, 148 mM Boric acid and adjust pH to 8.6) and nuclease-free water to a total of 20 µL reaction volume. Then 2 µL of 100% glycerol were added to stop the reaction for 10 minutes at room temperature. Purified RNA was precipitated with 100% isopropanol and Glycoblue overnight at -20°C. The pellet was washed once with 75% ethanol and dissolved in nuclease-free water. Then, RNA was size-selected between 18- to 30-nt on 15% TBE-urea gel. Purified fraction was confirmed by Bioanalyzer sRNA chip (Agilent). Small RNA library preparation was based on the NEBNext Multiplex sRNA Library Prep Set for Illumina (New England BioLabs) with slight modifications. To counteract ligation biases, the 3’ and 5' adapters contained 4 random bases at the 5’- and 3’-end, respectively, and were chemically synthesized by BioScientific. Adapter-ligated RNA was reverse-transcribed and PCR-amplified for 14 cycles using index primers. The PCR-amplified cDNA construct was purified using AMPure XP beads (Beckman Coulter). The purified PCR reaction was checked on the Bioanalyzer using High Sensitivity DNA chip (Agilent). Size selection of the sRNA library was done on LabChip XT instrument (Perkin Elmer) using DNA 300 assay kit. Only the fraction containing 140-165 bp was pooled in equal molar ratio. The resulting 10 nM pool was denatured to 10 pmol with 5% PhiX spike-in and sequenced as single-read on HiSeq 2500 (Illumina) in rapid mode for 51 cycles (plus 7 cycles index read)  using on-board cluster generation.
GEO Accession
GSM2771219