RNA-Seq of Homo sapiens: M-CSF induced iPSC, Donor3, WT, treated with Mtb infection for 24 hours
Low passage Mycobacterium tuberculosis (Mtb) strain Erdman was used for infected MCDM and AM-L macrophages. Mtb was grown to log phase in 7H9 Middlebrook medium supplemented with 0.5% glycerol, 0.05% Tween-80, and 10% oleic acid-albumin-dextrose-catalase (OADC) while maintained at 37. To prepare a single-cell mycobacterial suspension, Mtb was washed by centrifugation twice with phosphate buffered saline supplemented with 0.05% Tween-80 (PBST80), followed by centrifugation at 200 x g for 10 minutes to remove aggregated bacteria. Based on the OD600 of the final supernatant, a conversion factor of OD600 of 1.0 equates to approximately 5x108 CFU/mL was used to prepare the inoculum. Macrophages were infected at a target multiplicity of infection (MOI) of 3, unless stated otherwise. After approximately 4-hours, extracellular Mtb was removed by washing once with phosphate buffered saline (PBS). Phase separation in cells lysed in 1mL TRIzol Reagent was induced with 200 L chloroform. RNA was extracted from 350 L of the aqueous phase using the miRNeasy Micro Kit on the QIAcube Connect according to the manufacturers protocol. Samples were eluted in 34 L (11749) or 15 L (15727) RNase-free water. After RiboGreen quantification and quality control by Agilent BioAnalyzer, 29-100 ng of total RNA with RIN values of 6.5-9.8 underwent polyA selection and TruSeq library preparation according to instructions provided by Illumina, with 8 cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 in a PE50 run, using the NovaSeq 6000 SP Reagent Kit (100 Cycles).  An average of 59 million paired reads was generated per sample. Ribosomal reads represented 1.1-2% of the total reads generated and the percent of mRNA bases averaged 78%.
SUB15466177
Exp5_Donor3_170_WT_M-CSF_Mtb-infected-MOI1_24hrs_a