title: GSM4212279: RNA-seq rep 1 and 2; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: yeast RNA were extracted using Hot Acid Phenol Extraction method (Martine A. Collart and Salvatore Oliviero, 1993) Total cDNA was generated using anchored oligo-dT primer (5′-ACGACGCTCTTCCGATCTNNNNNNNN[10bp index]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′, where “N” is any base and “V” is either “A”, “C” or “G”; IDT) and SuperScript IV (Life Technologies) with the standard protocol. Second strand synthesis was carried out at 16°C for 180 min with NEBnext Second Strand Synthesis module (NEB) following the commercial protocol. cDNA was tagmented with Nextera tagmentation kit (Illumina) at 55°C for 5 min following commerical protocol. The reaction was stopped by adding 1x DNA binding buffer (Zymo) 6 and incubating at room temperature for 5 min. Each well was then purified using 1.5x AMPure XP beads (Beckman Coulter) following the commerical protocol and eluted in 16 μL of buffer EB (Qiagen). Each sample was then mixed with 2μL of 10 μM indexed P5 primers (5′- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC-3′; ID) and P7 primers (5′-CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG-3′, IDT) and 20 μL NEBNext High-Fidelity 2X PCR Master Mix (NEB). Amplification was carried out using the following program: 72°C for 5 min, 98°C for 30 sec, 10 cycles of (98°C for 10 sec, 66°C for 30 sec, 72°C for 1 min) and a final 72°C for 5 min. The library was purified with 0.8x AMPure XP beads (Beckman Coulter) and prepared for sequencing using Illumina Nextseq.
identifiers.namespace: GEO
identifiers.id: GSM4212279