title: RNA-Seq of Homo sapiens: GM-CSF induced iPSC, Donor3, Irg1KO, treated with media for 24 hours
design: 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, 2ng total RNA with RNA integrity numbers ranging from 4.1 to 10 underwent amplification using the SMART-Seq v4 Ultra Low Input RNA Kit, with 12 cycles of amplification. Subsequently, 7ng of amplified cDNA was used to prepare libraries with the KAPA Hyper Prep Kit using 8 cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 X in a PE100 run, using the NovaSeq 6000 S4 Reagent Kit (200 cycles).  An average of 34 reads million paired reads were generated per sample. Ribosomal reads represented 0.04-0.14% of the total reads generated and the percent of mRNA bases averaged 93%.
library_name: Donor3_170_Irg1KO_GM-CSF_uninfected_24hrs_b
identifiers.namespace: SUB15466177
identifiers.id: Donor3_170_Irg1KO_GM-CSF_uninfected_24hrs_b