Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Worms were lysed and monosomes purified (Hendriks & Gaidatzis et.al) and RNA was isolated using Tri-reagent (MRC) The RNA from the monosomal fraction was separated using a 15% TBE-Urea Gel (Invitrogen) and the region around 28-30 nucleotides excised to isolate Ribosome protected fragments (RPFs). The gel piece was forced through a pierced small tube inside an eppendorf tube by centrifugation and RNA from the gel debris was eluted by overnight incubation in 600 µl cracking buffer (20 mM Tris-HCl (pH 7.9), 1 mM EDTA, 400 mM NH4Acetate, 0.5 % SDS). RNA was precipitated with isopropanol at -80 °C for at least 4 hours (isopropanol precipitation). RPFs were 3’ dephosphorylated with 10 Units of T4 polynucleotide kinase (NEB) in T4 PNK buffer with 40 Units of RNasin for 1 hour at 37 °C. Following isopropanol precipitation, the RNA samples were ligated to 3’ adapters according to the Illumina® TruSeq™ Small RNA Sample Preparation protocol and using the reagents of the kit, then again precipitated with isopropanol. Ligation products were 5’ phosphorylated for 30 minutes at 37 °C with 15 Units of T4 polynucleotide kinase (NEB) in T4 PNK buffer, 1 mM ATP and 40 Units of RNasin. Following heat-inactivation of the enzyme for 10 minutes at 70 °C, the RNA was precipitated by isopropanol. Ligation to 5’ adapters, reverse transcription, PCR amplification with barcoded primers and gel-purification of the PCR products were performed using the Illumina® TruSeq™ Small RNA Sample Prep kit. Four different barcodes (RPIX 2, 4, 5, 6) were used.