On day 7, BMM were infected with Mtb H37Rv strain (MOI 5), followed by washing 2X with PBS. BMM were infected with H37Rv for 4, 8, 24, or 48 hours prior to RNA extraction.
Growth protocol
Bone marrow-derived macrophages (BMM) were cultured in complete RPMI (cRPMI; plus 10% FBS, 2 mM l-glutamine, penicillin and streptomycin) with recombinant human CSF-1 (50 ng/ml) for 6 days.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from cells using TRIzol (Invitrogen) according to manufacturer's instructions. Prior to labeling, sample integrity was checked using an Agilent 2100 Bioanalyzer.
Label
biotin
Label protocol
Total RNA samples were labeled as described in the Whole Transcript Sense Target Labeling protocol (Affymetrix)
Hybridization protocol
Samples were hybridized according to the Whole Transcript Sense Target Labeling protocol: Heat cocktail mix at 99°C for 5 minutes, 45°C for 5 minutes, and then centrifuge at 13,200g for 1 minute. Transfer 200uL of hyb solution to each array. Hybridize 16 hours at 45°C at 60rpm. Wash and stain using fluidics protocol FS450_0001.
Scan protocol
Affymetrix Gene ChIP Scanner 3000 7G
Description
naïve WT BMM
Data processing
Microarrays were normalized at the gene level using the BrainArray custom CDF (Entrez Gene, Version 14) for probeset definitions and RMA as implemented in the justRMA function of the Bioconductor package affy for background adjustment, quantile normalization and summarization. BrainArray version 14: MoEx10stv1_Mm_ENTREZG
