title: IP_PI_Extract_2
organism: Listeria monocytogenes EGD-e
description: Protocols: Briefly, each sample was incubated with a mix of 30 g of anti-Zea antibodies (10 g of each antibody) (overnight, 4 ˚C, shacking) or 30 g of normal rabbit IgG (CellSignaling) . Then, 50 L of Protein A sepharose were added for further 2 hours to recover immunocomplexes. The beads were washed four times with lysis buffer and treated with Turbo DNAse (Ambion) for 10 min at 37 ˚C in 200 L of 1X Turbo DNAse buffer. Samples were vortexed for 30 s after the addition of 200 L of acid phenol (Ambion). Then, 50 L of chloroform (Sigma) were added and samples were centrifuged for 5 min at 10 000 × g at 4 ˚C. Aqueous upper phase was recovered and transferred to a new Eppendorf tube. L. monocytogenes growth culture (50 mL) at the stationary phase (OD600nm = 3.5) was centrifuged (2862 × g, 20 min, 4 ˚C). Culture medium (10 mL) was filtered (0.22 M) and concentrated to 1 mL by using Amicon centrifugal filter unit (3K cut-off). Concentrated medium was then supplemented with 0,05% of TX100, centrifuged again (18407 × g, 20 min, 4 ˚C) and left on ice while preparing bacterial cytosolic extracts. Bacterial pellet was washed thrice in ice-cold PBS and lysed by mechanical shaking in a FastPrep apparatus (described above) in 1 mL of lysis buffer (150mM KCl, 1mM DTT, 25mM Tris pH7.4, 0.05% TX100) supplemented with protease inhibitors mixture. Bacterial cytosol was recovered by centrifugation (two serial centrifugations at 18407 × g, 20 min, 4 ˚C) and protein concentration determine by Bradford assay. 1 mL of bacterial cytosol and concentrated culture medium (corresponding to 50 mL and 10 mL of the bacterial cytosol and culture medium, respectively) were individually loaded on a Superose 6 10/300 GL column, which was previously equilibrated with lysis buffer without TX100. About 161 fractions of 220 L were collected, and one fraction every seven fractions was concentrated by acetone precipitation and the presence of Zea and EF-Tu analyzed by Western blotting after wet transfer onto nitrocellulose membrane. Fractions containing the complexes (A or B) were next pooled and processed for immunoprecipitation assays. RNA was precipitated by adding the same volume of isopropanol and 0.3M of sodium acetate (Ambion) and 1 L of glycogen (Invitrogen). Samples were centrifuged (30 min, 10 000 × g, 4 ˚C), and RNA was washed once with 70% ethanol before being resuspended in 25 L of nuclease-free water. RNA was analyzed by Bioanalyser RNA pico kit (Agilent). Purified RNA was fragmented with RNA fragmentation reagents (Thermofisher), purified by ethanol precipitation and quality-controlled by Bioanalyser. Directional RNA-seq libraries were prepared with 30 ng of purified RNA for each sample by using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) according to the manufacturer's instructions. When required, the RNA samples were spike-in with a synthetic in vitro transcribed AdML splicing reporter(Allemand et al., 2016) in order to have 30 ng of total RNA.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-10-16T04:05:38Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-02-11T16:07:24Z
attributes.tag: External Id
attributes.value: SAMEA5332380
attributes.tag: INSDC center name
attributes.value: Institut Pasteur - Hub Bioinformatique et Biostatistique - Centre de Bioinformatique, Biostatistique et Biologie Integrative (C3BI, USR 3756 IP CNRS) - Paris, France
attributes.tag: INSDC first public
attributes.value: 2019-10-16T04:05:38Z
attributes.tag: INSDC last update
attributes.value: 2019-02-11T16:07:24Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7665:IP_PI_Extract_2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: growth condition
attributes.value: Stationnary phase
attributes.tag: sample name
attributes.value: E-MTAB-7665:IP_PI_Extract_2
attributes.tag: scientific_name
attributes.value: Listeria monocytogenes EGD-e
attributes.tag: strain
attributes.value: EGD-e WT