title: GSM1863249: exo2_1_NET_Seq_IN; Schizosaccharomyces pombe; OTHER
library_construction_protocol: NET-Seq analysis was performed from two biological replicates of strains YAM2492 (Rpb3-flag) and YAM2507 (exo2-delta Rpb3-flag) of S. pombe. Libraries were constructed according previously published protocol (Churchman and Weissman, 2012), with minor modifications, starting from 1L of exponentially growing cells. Ligation of DNA 3â€™-linker was performed as described (Marquardt et al., 2014), starting with 2-3 µg of purified nascent RNA. Ligated nascent RNA was then submitted to alkaline fragmentation for 20 minutes at 95°C. Single-end sequencing (50 nt) of the libraries was performed on a HiSeq 2500 sequencer, giving between 20 and 50 million reads. NET-Seq is a sequencing of 3' end of nascent transcript produced by RNA Polymerase II. In this protocol, transcribing RNA Pol II is isolated, then nascent transcripts are purified and used to construct libraries, using a non-standard protocol described by Weismann's lab. These samples are called "IP" (as for ChIP). "Input" are control libraries constructed from the same biological samples, using the very same control but without purification of RNA Pol II. So they are similar to "total" RNA libraries, but constructed using the same protocol as IP, for comparison.