GSM2069584: BY4741_NAI_1; Saccharomyces cerevisiae; OTHER
Cells were pelleted and total RNA was extracted using Gunanidium thiocyanate and phenol-chloroform method as previously described (Tollervey 1987, EMBO Journal). Ribosomal RNA was depleted usign the Illumina RiboZero kit for yeast, according to the manufactuer's procedures. cDNA libraries were generated from purified ribosomal RNA using an RT oligonucteotide containing random hexamers (SuperScript III, Invitrogen). 5' adapters were ligated on (CircLigase II, EpiCentre) and 3' adaptors generated during PCR amplification. Random primer used: 5’- CGTGTGCTCTTCCGATCT NNN NNN-3’ reverse transcription primer: 5'-AGATCGGAAGAGCACACG-3' Adapters ligated to cDNAs: P5_RAND_Barcode_IDX1_phospho_adapter: 5’P-N  cgt gat NNN NNN AGA TCG GAA GAG CGT CGT GTA GGG -ddC-3’ Adapters ligated to cDNAs: P5_RAND_Barcode_IDX2_phospho_adapter: 5’P-N aca tcg NNN NNN AGA TCG GAA GAG CGT CGT GTA GGG -ddC -3’ Adapters ligated to cDNAs: P5_RAND_Barcode_IDX3_phospho_adapter: 5’P-N gcc taa NNN NNN AGA TCG GAA GAG CGT CGT GTA GGG -ddC -3’ Adapters ligated to cDNAs: P5_RAND_Barcode_IDX4_phospho_adapter: 5’P-N tgg tca NNN NNN AGA TCG GAA GAG CGT CGT GTA GGG -ddC -3’ PCR oligonucleotides: P5_Forward: 5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’ PCR oligonucleotides: BC1: 5′-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' PCR oligonucleotides: BC6: 5′-CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′ All libraries were 50bp paired-end sequenced on a HiSeq 2500