GSM1541705: by_poly_1_p; Saccharomyces cerevisiae; RNA-Seq
All  samples were collected by centrifugation and frozen in liquid N2. Total RNA was isolated by a standard hot phenol method and treated with RNase-free DNaseI using Turbo DNA-free kit (Ambion) We used 10-100 µg of DNA-free total RNA as starting material. As an internal control, we added polyadenylated in vitro transcripts (ATCC 87482, 87483 and 87484, that were modified to be enriched respectively in 5’cap, 5’PPP or 5’P ends) and S. pombe RNA. For 5’P sequencing (5PSeq), samples were directly subjected to RNA ligation. To generate a negative control of 5’P molecules (5PSeq-fragmented) RNA sample was chemically fragmented by incubating the samples at 80ºC for 5 min in the presence of RNA fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM KOAc, 30 mM MgOAc). Fragmented 5’OH sites were then phosphorilated by treatment with 5 unit of T4 Polynucleotide Kinase (T4 PNK, NEB) for 1 hour at 37ºC in the presence of RNase inhibitor (RNasin +, Promega). The sample was phenol:chloroform purified and ethanol precipitated and subjected to RNA ligation. From the ligation step we used the same protocol all the methods. The treated RNA sample was incubated overnight at 16ºC with 20 units of T4 RNA ligase 1 (NEB) in the presence of 10 mM DNA/RNA rP5_RND oligo (CTTTCCCTACACGACGCTCTTCCGATrCrUrNrNrNrNrNrNrNrN). RNA integrity was controlled by RNA Bioanalyzer and then polyA purified using Oligotex (Quiagen) according to manufacturer’s instructions. PolyA enriched RNA was fragmented at 80ºC for 5 min in the presence of RNA fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM KOAc, 30 mM MgOAc). RNA was retrotranscripted with Superscript II (Life Technologies) and primed with random hexamers. The retrotranscription reaction was incubated for 10 minutes at 25ºC, 5 minutes at 42ºC and heat inactivated for 15 minutes at 72ºC. Second cDNA strand was perform by a unique cycle of PCR (1 min at 98ºC; 2 min at 50ºC and 15 at 72ºC) using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) and priming with BioNotI-P5-PET (Biotin-TATAGCGGCCGCAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT). From this point libraries were generated as previously described (PMID:23295673). Briefly double stranded cDNA was first purified using HighPrep beads (Magbio) and then bound to Dynabeads M-280 Streptavidin beads (Life Technologies) according to manufacturers instructions. Bound DNA molecules were successively washed and subjected to Ends repair, A addition and adaptor ligation using Nebnext DNA Library Prep Master Mix (NEB). 0.5µL of common adapter (2.5µM) was ligated to each sample. The common adapter was prepared by annealing P7MPX_linker_for and P7MPX_linker_rev. Beads were washed and subjected to PCR amplification (30s 96ºC; 15-18 cycles (30s 96ºC; 30s 65ºC; 30s 72ºC); 5min 72ºC) using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) and 0.1µM final of PE1.0 and the appropriate indexed PE2_MTX PCR oligo. Sample was size selected using 0.6x-0.9x (v/v) HighPrep beads (Magbio) to 300-500 bp. Libraries were sequenced (single-end) using either an Illumina HiSeq 2000 or a MiSeq. The first experiments were indexed adding a barcoded P7 linker (as in PMID:23295673) and subjected to a paired-end sequencing where first read contains the 8nt molecular barcode followed by the 5’sequence and the reverse read contains a 6 nt index followed by and A and the reverse complementary sequence of the identified transcripts.