title: GSM3018357: CoIP_Cas9 chromosomally Flag tagged (positive) OD600 0.5; Neisseria meningitidis 8013; RIP-Seq
library_construction_protocol: Samples for RNA-Seq were collected from bacterial cul- tures grown to optical densities (OD)600 of 0.5 or 1.5, corre- sponding to the mid logarithmic and late logarithmic/early stationary growth phase, respectively. Samples were fixated by the addition of STOP Mix [95% (vol/vol) EtOH and 5% (vol/vol) phenol], frozen in liquid nitrogen and subsequently stored at−80◦C untilRNA preparation. For RNA extraction frozen bacterial cultureswere thawed on ice, cen- trifuged and cell pellets were resuspended in a lysis solution consisting of 800 ?l of 0.5 mg/ml lysozyme in Tris-EDTA (TE) buffer (pH 8.0) and 80 ?l 10% sodium dodecyl sul- phate (SDS). Bacterial cells were lysed by placing the sam- ples for 1–2 min at 65◦C in a water bath. Total RNA was extracted fromthe lysates using the hot phenol method. N. meningitidis expressing either 3× FLAG-tagged- or wild-type (WT) Cas9 protein were grown in the presence of kanamycin until an OD600 of 0.5 and 1.5, respectively. For each strain cells equivalent to an OD600 of 50 were collected and subjected to Cas9 coIP and control coIP as described in detail in ref.14 and briefly in the Supplementary Materials and Methods. cDNA libraries of RNA-seq and RIP-seq samples were constructed by Vertis Biotechnology AG, Munich, Germany. cDNA libraries were pooled and sequenced using a MiSeqmachine  in the single-read mode. RNA-seq with wild type and Cas9 mutant and RIP-seq RNA co-immunoprecipitation (coIP) with Cas9
attributes.tag: GEO Accession
attributes.value: GSM3018357