GSM3048802: TAP rep3; Caenorhabditis elegans; ncRNA-Seq
Gravid adults were washed off plate with M9 buffer and lysed in 250 µL of Worm Lysis Buffer (0.2M NaCl, 0.1M Tris pH 8.5, 50 mM EDTA, 0.5% SDS) with 150 µg Proteinase K (Sigma-Aldrich #P2308) for at least 90 minutes at 65oC. 750 uL of Trizol LS were added per sample and extraction followed manufacturer's instructions, with the exception that phase-lock tubes were used to facilitate phase-separation. Total RNA extraction was followed by TURBO DNase treatment (Life Technologies #AM1907) according to manufacturer's instructions. Small RNAs were isolated from DNase-treated total RNA using MirVana (Life Technologies #AM1561). A single RNA sample was divided into 6 technical replicates. Three of these technical replicates were treated with Tobacco Acid Phosphatase (TAP, Epicentre) and the other three, with RNA 5'Pyrophosphohydrolase (RppH, New England Biolabs, #M0356S). For TAP-treated samples, 1 µg of total RNA was incubated for 2 hours at 37C, with 5 units of TAP enzyme and 10X TAP buffer. For RppH treated samples, 1µg of RNA was incubated for 1 hour at 37°C, with 5 units of RppH enzyme and 10X NEB Buffer 2. To stop the RppH treatment, 500mM EDTA was added and samples were incubated for 5 minutes at 65°C. Small RNAs (16-30nt) were enriched by performing size selection of the RNA prior to library preparation. RNA samples were run on a 15% TBE-Urea polyacrylamide gel (BioRad), and purified with sodium chloride/isopropanol precipitation. Library preparation was performed using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England BioLabs) as recommended by the manufacturer (protocol version v2.0 8/13), but replacing the RNA ligation adapters with adapters with 4 additional random bases at their 3' ends to allow identification of PCR clonal artefacts (synthesized by Bioo Scientific). Fourteen PCR cycles were used for library amplification The PCR-amplified cDNA was purified using AMPure XP beads (Beckman Coulter). Size selection of the small RNA library was done on LabChip XT instrument (Perkin Elmer) using DNA 300 assay kit extracting the 135-170 bp fraction. The 135-170 bp library fractions of the RppH and TAP-treated libraries were pooled in equal molar ratio. The resulting 4 nM pool was denatured and diluted to 9 pM with 5% PhiX spike-in and sequenced as single-read on MiSeq (Illumina) for 68 cycles. Libraries for an untreated sample, originating from a different biological replicate, were also prepared using the protocol above, but were sequenced as single-read on HiSeq 2500 (Illumina).
GEO Accession
GSM3048802