GSM3504842: C. elegans RDE-3b #5; Saccharomyces cerevisiae BY4741; RNA-Seq
RNA was extracted from 25 OD of yeast corresponding to each sample by modification of a previously described method.  Briefly, to each sample, 0.5 g of 0.5 mm acid washed beads (Sigma-Aldrich), 0.5 mL of RNA ISO buffer (500 mM NaCl, 200 mM Tris-Cl pH 7.5, 10 mM EDTA, 1 % SDS) and 0.5 mL of phenol-chloroform-isoamyl alcohol pH 6.7 (PCA, Fisher Scientific) was added.  Samples were lysed with 10 cycles that each consisted of vortexing for 20 seconds and incubation on ice for 30 seconds.  1.5 volumes (relative to starting amount of ISO Buffer) of RNA ISO Buffer and of PCA were added, and samples were centrifuged at 4°C to separate phases. The aqueous layer was transferred to a pre-spun phase-lock gel (heavy) tube (5PRIME/Quantabio); an equal volume of PCA was added and mixed prior to centrifugation at room temperature to separate phases.  The aqueous layer was transferred to 2 new tubes for ethanol precipitation with 2 volumes of 100% ethanol followed incubation at -80°C for 1 hour to overnight. Precipitated RNA was pelleted by centrifugation at 4°C.  Each pellet was dissolved in 25 μL nuclease-free water and combined into 1 tube per sample.  Co-purifying DNA was digested with 20 U of Turbo DNase (Invitrogen) at 37°C for 4 hours, and RNA was cleaned up with the GeneJET RNA Purification Kit (Thermo Scientific), and eluted with 50 μL of DEPC-treated water. Total RNA (100 ng) was ligated with 20 pmol of a 5ʹ adenylated primer containing a 7-nucleotide random DNA sequence (random heptamer), Illumina TruSeq adapter sequence and a 3ʹ dideoxycytidine (5ʹ-A(pp) NNNNNNN TGGAATTCTCGGGTGCCAAGG ddC-3ʹ) using 200 U of T4 RNA ligase 2, truncated KQ (New England BioLabs) in a 20μL reaction with 16°C overnight incubation.  This ligation added the random heptamer and Illumina TruSeq adapter sequence to the 3ʹ end of the RNAs in the sample.  Half of the ligation reaction (10 μL) was reverse transcribed using 5 pmol of Illumina RNA RT primer (5ʹ-GCCTTGGCACCCGAGAATTCCA-3ʹ) and ImProm-II Reverse Transcriptase (Promega Corporation) with 1.5 mM MgCl2 and 0.5 mM dNTPs, according to manufacturer's instructions.  Samples were then PCR-amplified with a forward primer consisting of Illumina-specific sequences and sequence (underlined),  specific to the tRNA reporter   (5ʹ-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATCGAGGATCACCCATGTCGCAG-3ʹ) and a reverse Illumina RNA PCR Primer with various indices used for multiplexing, using GoTaq Green PCR Master Mix (Promega Corporation). PCR products were run on an 8% polyacrylamide 8M urea gel and gel extracted. Resulting samples for each sequencing run were combined in equimolar amounts and run on an Illumina HiSeq2000 or HiSeq2500 (2x50 bp or 2x100 bp), to produce approximately 1 x 106 reads per sample.
GEO Accession
GSM3504842