Nanodrop 8000 instrument

RPE-1 cells (ATCC) were seeded in a six-well plate, silenced for 96 hr, washed twice in PBS, and resuspended in 300 μl of Tri-Reagent (MilliporeSigma). RNA was isolated by centrifugation followed by chloroform extraction, isopropanol precipitation, washing twice in 75% ethanol, and resuspended in 20 μl DEPC-treated water (Thermo Fisher Scientific). Total RNA was quantified with a Nanodrop 8000 Instrument (Thermo Fisher Scientific). 1 μg of total RNA was used for the reverse transcription reaction performed by High-Capacity RNA-to-cDNA Kit (Applied Biosystems), according to the manufacturer’s recommendations, in a 2× RT buffer mix, supplemented with dNTPs, random primers, and RT enzyme in a final volume of 20 μl. Quantitative real-time PCR (qRT-PCR) was performed using the comparative CT method and a Step-One-Plus Real-Time PCR Instrument (Thermo Fisher). Amplification of the 16 ng of cDNA was done in triplicate with TaqMan Universal PCR Master Mix (Thermo Fisher) for ADSL and GAPDH.

Transfected AD-293 cells were collected after two cold PBS washes by scraping into Tri-Reagent. RNA was isolated by chloroform extraction followed by centrifugation, isopropanol precipitation, 2 washes in 75% ethanol and resuspension in DEPC-treated water. Nucleic acid quantification was performed with a Nanodrop 8000 Instrument (ThermoFisher Scientific). cDNA was generated using 0.5–1 μg of total RNA and a High Capacity RNA-to-cDNA Kit (Applied Biosystems). Quantitative real-time PCR was performed using the comparative CT method and a StepOne Real-Time PCR System (Applied Biosystems). Amplification was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) or TaqMan Universal PCR Master Mix (Applied Biosystems). All assays were performed in duplicate. For TaqMan assays, mActB probe was used as an endogenous control for normalization and a specific Taqman probe was used for mouse or human TP73 (Hs01056231_m1), CCNO (Hs004389588_91), FOXJ1 (Hs00230964_m1), and TP73 (Hs01056231_m1). Primers used for SYBR Green assays (Sigma-Aldrich) can be found in Table S4.

Cryopreserved PBMCs (n=115) were thawed and resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 50 ng/mL recombinant human monocyte colony-stimulated factor (M-CSF) for 24h. CD14⁺ monocytes were enriched from PBMCs by magnetic separation (Monocyte Isolation Kit II, Miltenyi Biotec) and incubated in RPMI-10 with M-CSF for 24h. Monocytes were then infected with H37Rv Mtb at a multiplicity of infection (MOI) of 1 or media alone (uninfected control) and incubated at 37°C with 5% CO² for 6h. After incubation, both the media-only and Mtb-infected monocytes were lysed in TRIzol (Invitrogen), and RNA was isolated using the RNeasy Mini Kit according to the manufacturers’ instructions (Qiagen). The RNA samples were quantified using Nanodrop 8000 instrument (Thermo Scientific), and their quality was measured by TapeStation (Agilent) (RNA Integrity Number ≥ 8.0). Next, cDNA libraries were prepared with random hexanucleotide primers and rRNA depletion using SMARTer RNAseq Kit (Takara), followed by sequencing on Illumina HiSeq 2500 and Novaseq 6000 platforms as previously described (15 (link)).