organism: Mycobacterium tuberculosis variant bovis BCG
description: Protocols: Human monocyte THP-1 cells were differentiated into macrophage-like cells with 200 nM PMA for 24h, followed by 48h recovery in PMA-free media. Exponential phase M. bovis BCG Pasteur-GFP was washed in phosphate-buffered saline and re-suspended in RPMI without antibiotics. The inoculum was needle homogenised fifteen times to create a single-cell suspension. THP-1 cells were infected at an MOI of 10:1 for 4h at 37ºC with 5% CO2. After 4h, the cells were washed with PBS and then incubated in RPMI without antibiotics for a further 20h. Mycobacterial RNA was harvested using the GTC/Trizol differential lysis method and purified using RNeasy columns (Qiagen) with DNase treatment. Mycobacterial RNA yield and quality was assessed using the NanoDrop One Spectrophotometer (Thermo Scientific) and Agilent 4200 TapeStation (Agilent Technologies). Libraries for RNA sequencing were prepared from three (2ug total RNA) biological replicates of intracellular M. bovis BCG after rRNA depletion (Ribo-Zero, Illumina) using the NEBNext Ultra II kits (New England Biolabs), and sequenced on an Illumina NextSeq500 sequencer.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-01-03T15:59:21Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-06-14T10:15:20Z
attributes.tag: External Id
attributes.value: SAMEA5717414
attributes.tag: INSDC center name
attributes.value: Brighton and Sussex Medical School, University of Sussex, UK
attributes.tag: INSDC first public
attributes.value: 2020-01-03T15:59:21Z
attributes.tag: INSDC last update
attributes.value: 2019-06-14T10:15:20Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8057:Intracellular BCG 6_4_D
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: growth condition
attributes.value: grown in human macrophage-like THP-1 cells
attributes.tag: sample name
attributes.value: E-MTAB-8057:Intracellular BCG 6_4_D
attributes.tag: sampling time
attributes.value: 24
attributes.tag: scientific_name
attributes.value: Mycobacterium tuberculosis variant bovis BCG
attributes.tag: strain
attributes.value: Pasteur 1173P2