title: GSM1467476: rrp6Δ; Schizosaccharomyces pombe; RNA-Seq
library_construction_protocol: Hot phenol RNA dextraction protocol (Schmitt ME, Brown TA & Trumpower BL (1990) A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 18: 3091–3092) PNK treated total RNA was purified with the RNA clean and concentrator kit (Zymo Research). 120 pmol of 5' phosphorylated 3' RACE adapter oligonucleotide (cctatagtgagtcgtattaattctgtgctcgc(tdd)) were mixed with 3 µg of purified RNA and ligated with T4 RNA ligase as above. 1 µg of ligated RNA was reverse-transcribed with Superscript III (Invitrogen) using adapter-specific oligonucleotide (GCGAGCACAGAATTAATACGACT). cDNA was PCR-amplified with the Phusion High-Fidelity PCR kit (Thermo Scientific) and PCR products were sequenced. Primers to amlify U6 snRNA (acactctttccctacacgacgctcttccgatctgatcttcggatcactttggt + ctcggcattcctgctgaaccgctcttccgatctcgcggatccgaattaatacgactcactatagg); Primers to amplify tagged U6 (acactctttccctacacgacgctcttccgatctgatcttcggatcactttgatcttcg + ctcggcattcctgctgaaccgctcttccgatctcgcggatccgaattaatacgactcactatagg)