title: GSM2258031: NETseq_Spt5_T0_II; Schizosaccharomyces pombe; OTHER
library_construction_protocol: One liter cultures of S. pombe at 10^7 cells/ml were spiked-in with S. cerevisiae (10% by cell number), and the cells were collected by filtration. One of the libraries used for our analysis (T0_I, before depletion of Spt5) did not have S. cerevisiae as spike-in. Cells were flash frozen by forcing the cells through a syringe into liquid nitrogen. The frozen cells were mixed with 4 ml of frozen (by dropping 50 µl drops into liquid nitrogen) lysis-buffer without MnCl2 and protease inhibitors   and lysed in a mixer-mill using 8 cycles at 15 Hz (Lysis buffer: 20mM HEPES, 110mM KOAc, 0.5% Triton X-100, 0.1% Tween 20, 10mM MnCl2, 1X protease inhibitors Roche EDTA-free). This lysate was thawed on ice and 1 ml lysis buffer with 50mM MnCl2 and 5x protease inhibitor, was added to the lysate along with 660 µl of DNaseI (RQ1, Promega) and incubated on ice for 30 minutes. Solubilized RNAPII was then immunoprecipitated along with the associated RNA using  FLAG M2 beads (SIGMA), and eluted using 3X-FLAG peptide (SIGMA). RNA was extracted from the eluate using Qiagen miRNEasy kit. Library prep was performed as previously described (PMID: 21248844) except that a modified linker-1 with a randomized hexamer was used for all libraries from experiment 2.
attributes.tag: GEO Accession
attributes.value: GSM2258031