title: Cherry_E_1_1
organism: Listeria monocytogenes EGD-e
description: Protocols: Four 15 cm2 tissue culture dishes per cell line were pretreated with 0.1mg/mL poly-L-Lysine-Hydrobromide (Sigma), rinsed with distilled water and let dry 1 hour before plating the cells. 30-40.10ˆ6 cells were plated per dish in 20 mL of DMEM medium for 24 hours before infection. Overnight L. monocytogenes EGD-e cultures in BHI were diluted 1/20 the day of infection in fresh BHI and grown up to OD600nm = 1. Medium was then recovered by centrifugation (2862 × g, 20 min, 4 ˚C) and filtered (0.22 M). Bacterial pellet was stored at -80 ˚C for subsequent RNA extraction. 10 mL of filtered culture medium were desalted and RNA was extracted. Each plate is infected with an MOI of 50 for 1 hour before replacing the media with complete DMEM containing 10 μg/mL of gentamicin to kill extracellular bacteria. After additional 3 hours (in total 4 hours of infection), plates were rinsed twice with ice-cold PBS, crosslinked at 400 mJ/cm2 in 10 mL of ice-cold PBS/plate and cells were then scraped, pelleted and resuspended in 8 mL of MOPS lysis buffer (20 mM MOPS-KOH pH 7.4, 120 mM KCl, 0.5% Igepal, 2 mM beta-mercaptoethanol, supplemented with protease inhibitors mixture and RNasin at 12,5 U/l and protease inhibitors mixture (Roche) and incubated for 20 min on ice. 100 μL/dish of Streptactin Sepharose beads (GE Healthcare) was washed in MOPS washing buffer (20 mM MOPS-KOH pH 7.4, 120 mM KCl, 2mM beta-mercaptoethanol, supplemented with RNasin 12,5 U/L and protease inhibitors mixture and finally resuspended in 1 mL of MOPS lysis buffer per initial culture dish. Ribonucleoprotein extract was incubated with Streptactin beads for 2 hours at 4 °C. The beads were washed three times with MOPS washing buffer and centrifuged at 1600 × g, 5 min at 4°C. Strep-tagged proteins are then eluted twice for 15 min at 4 °C in 250 μL of 1X elution buffer (IBA, Biotin Elution Buffer 10X). The sample is then treated with proteinase K (Roche) in v/v of 2X proteinase K buffer (200 mM Tris pH 8, 100 mM NaCl, 20 mM EDTA, Urea 4M) for 20 min at 4 °C that has been preincubated 20 min at 37 °C to remove RNase contamination. RNA purification was performed using TRI Reagent LS (Sigma). RNA was dissolved in 50 μL of DNase-free and RNase-free ultrapure water. Directional RNA-seq libraries were prepared with 100 ng of purified RNA for each sample by using NEBNext Multiplex Small RNA Library Prep Set for Illumina according to the manufacturer's instructions.
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: SAMEA5332363
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:Cherry_E_1_1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: growth condition
attributes.value: Stationnary phase
attributes.tag: sample name
attributes.value: E-MTAB-7665:Cherry_E_1_1
attributes.tag: scientific_name
attributes.value: Listeria monocytogenes EGD-e
attributes.tag: strain
attributes.value: EGD-e WT