title: GSM2805395: nRNA_37C_prp2-1; Schizosaccharomyces pombe; RNA-Seq
library_construction_protocol: Nascent RNA was prepared using a nascent RNA isolation protocol for yeast (Carrillo Oesterreich et al. 2010) and Phenol:Chloroform:IAA, 25:24:1, pH 6.6 extraction. PolyA- RNA was obtained using oligo-dT coated magnetic beads binding to polyA+ RNA (Dynabeads mRNA DIRECT Micro Purification Kit, Life technologies). rRNA was removed using the Ribo-Zero Gold rRNA Removal Kit (Yeast, Epicenter/Illumina) from ~5 μg RNA. Nascent RNA was 3’ end ligated to the DNA adaptor (/5rApp/NNNNNCTGTAGGCACCATCAAT/3ddC/) and reverse transcribed into cDNA with a slight modification of the SMARTer PCR cDNA Synthesis Kit (Clontech). A custom primer reverse-complement to the 3’ end adaptor was used (AAGCAGTGGTATCAACGCAGAGTACATTGATGGTGCCTACAG). Maximal 1 µg of RNA was used per reaction. The subsequent low cycle PCR was carried out with the Advantage 2 PCR Kit according to the instructions provided by the kit. Double-stranded cDNA was further used for Pacific Biosciences library preparation and sequencing with standard protocols and kit from Pacific Biosciences (SMRTbell™ Template Prep Kit 1.0). SMRTbell™ Template Prep Kit 1.0
attributes.tag: GEO Accession
attributes.value: GSM2805395