Abi q6 instrument

Using an RNAprep Pure tissue kit (Tiangen, Beijing, China), total RNA was extracted from mouse tissues. A total of 2 μg of total RNA was reverse transcribed into cDNA with FastKing gDNA Dispelling RT SuperMix (Tiangen, Beijing, China). For RNA quantification, SYBR Green qPCR SuperMix (Transgen Biotech, Beijing, China) was performed using an ABI Q6 instrument (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. The mRNA expression levels were normalized to Gapdh expression by the 2^−ΔΔCt value method. Information on primer sequences is listed in Supplementary Table S2.

We had extracted Genomic DNA from 200 μL of blood collected from each participant using a TIANamp Blood DNA Kit (Tiangen, Beijing city, China) and we followed the specific procedures in the literature (35 (link)–37 (link)). The extracted DNA is placed in a −80°C refrigerator until use. The allele-specific probes were purchased from Applied Biosystems. TaqMan real-time polymerase chain reaction of the samples is performed in 384-well plate with an ABI Q6 instrument (Thermo Fisher Scientific, Waltham, MA, United States) to genotype rs7404339 polymorphism in CDH5 (38 (link), 39 (link)). For quality control, each 384-well plate contained eight samples without DNA but with the same amount of distilled water. Moreover, to ensure the quality and accuracy of the genotyping results, we randomly selected 10% of the samples for a repeat analysis, and the results were 100% concordant.

Total RNA was isolated using RNAiso plus (9109; TaKaRa) according to the manufacturer’s instructions and subjected to reverse transcription with a PrimeScript RT reagent kit (RR037A; TaKaRa). RNA from M. tuberculosis clinical isolates was extracted via ultrasonication for 10 min (on/off time, 5 s/5 s) before adding RNAiso plus. All gene transcripts were quantified by real-time PCR with PowerUP SYBR green master mix (A25742; Applied Biosystems) using an ABI Q6 instrument (Applied Biosystems). GAPDH was used as a reference in RAW64.7 cells, and SigA was used as an internal control for the normalization in M. tuberculosis. The primers were used for qPCR as shown in Table S3.

RAW264.7 cells were infected with MS_WT or MS_Rv0790c at an MOI of 10 for 24 h. The cells were harvested for RT-PCR and the culture supernatants were harvested for ELISA. Total cellular RNA was extracted using TRIzol reagent (Invitrogen), according to the manufacturer’s protocol. cDNA was synthesized using a reverse transcription kit (Takara). The relative mRNA levels of cytokines were detected by RT-PCR amplification using an ABI Q6 instrument (Applied Biosystems), and the following specific primer pairs were used: GAPDH (sense- 5′-GAGCCAAACGGGTCATCA TCT-3′; antisense- 5′-GAGGGGCCATCCACAGTCTT-3′, TNF-α (sense- 5′-TCTTCT CGAACCCCGAGTGA-3′; antisense- 5′-CCTCTGATGGCACCACCA-3′), IL-6 (sense-5′-AGGAGACTTGCCTGGTGAAA-3′; antisense-5′-CAGGGGTGGTTATT G CATCT-3′). Gene expression was analyzed using the 2^−△△CT method (Chiang et al., 2012 (link); Salvador-Martin et al., 2020 (link)). TNF-α and IL-6 protein levels were determined using ELISA kits (Invitrogen), following the manufacturer’s protocols.

We extracted the DNA from the blood samples. The extracted DNA was placed in a −80°C refrigerator until use. For genotyping of the rs2910164 C>G polymorphism, we used ABI Q6 instrument (Applied Biosystems) and TaqMan assays [18–20 (link)]. The genotypes of cases and controls were determined during this process. For quality control, each 384-well plate contained eight samples without DNA but with the same amount of distilled water.

Total RNA was extracted using an RNAprep Pure tissue kit or RNAprep Pure cell kit (Tiangen, Beijing, China). The total RNA (2 μg) was then reverse transcribed to cDNA using the FastKing gDNA Dispelling RT SuperMix (Tiangen, China). Quantitative real-time PCR was performed in triplicate using the SYBR Green qPCR SuperMix (Transgen Biotech, Beijing, China) on an ABI Q6 instrument (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. Gene expression was normalized against the murine or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene. The primers used are listed in Supplementary Table S2.