description: Protocols: Bone marrow cells from femurs and tibia of mice were isolated and plated in dishes (not treated for tissue culture) containing high glucose DMEM (Life Technologies) with 5% horse serum (Sigma), 10% fetal bovine serum (Sigma), Penicillin-Streptomycin (Sigma), 10mM HEPES (Corning), 1% L-glutamine, 1% sodium pyruvate and Macrophage Colony-Stimulating Factor (Biolegend) at 10 ng/ml for 7 days prior to experimental procedure. For stimulation experiments, macrophages were collected in DMEM by cell scraper and reseeded in 6- or 12-well plates in DMEM containing 10% Fetal bovine serum, Penicillin-Streptomycin (Sigma), 10mM HEPES (Corning) and allowed to adhere. After 24 hours, cells were washed with sterile PBS and resuspended in fresh media and stimulated with live D hansenii (MOI- 1:10) or vehicle (PBS). 24 hours post-stimulation cells were lysed and subjected to RNA extraction. Total RNA was isolated from whole cell lysates with Trizol Reagent via NucleoSpin RNA Isolation Kitswith Trizol Reagent according to the manufacturer's protocol. RNA was DNase-treated and purified via Qiagen RNA Purification columns according to the manufacturer's instructions. Total RNA concentration and purity was obtained from an absorbance ratio at 260nm and 280nm. Total RNA quality was then determined by Agilent 2100 bioanalyzer (Agilent Technologies) according to manufacturer's recommendations. RNA library preparations and sequencing reactions were conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA). RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit using manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligo d(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on a single lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to manufacturer's instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.
attributes.tag: ENA first public
attributes.value: 2021-04-14
attributes.tag: ENA last update
attributes.value: 2020-11-25
attributes.tag: External Id
attributes.value: SAMEA7624921
attributes.tag: INSDC center alias
attributes.value: Washington University St Louis
attributes.tag: INSDC center name
attributes.value: Washington University St Louis
attributes.tag: INSDC first public
attributes.value: 2021-04-14T00:22:17Z
attributes.tag: INSDC last update
attributes.value: 2020-11-25T13:54:21Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9806:6hr.A
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: macrophage
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: bone marrow
attributes.tag: sample name
attributes.value: E-MTAB-9806:6hr.A
attributes.tag: strain
attributes.value: C57BL/6