Nanodrop 2000 platform

Total RNA was extracted from cells or tissues with TRIzol reagent (Omega, Norcross, GA, USA) according to the manufacturer’s instructions. RNA quality and quantity were assessed with the NanoDrop2000 platform (Thermo Fisher Scientific, Waltham, MA, USA). cDNA Synthesis kits (TaKaRa, Otsu, Japan) were used for mRNA reverse transcription. Real-time PCR analysis was performed with SYBR Green PCR Kits (TaKaRa) on the 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The amplified transcript levels of genes were normalized to β-actin with the optimized comparative 2^−ΔΔCt value method. The primer sequences are in Additional file 1: Table S1.

Genomic DNA was extracted from whole blood samples with GoldMag whole blood genomic DNA purification kit (GoldMag Co. Ltd. Xi'an city, China). DNA concentration was evaluated with a NanoDrop 2000 platform (Thermo Fisher S 95% CIentific, Waltham, MA, USA), according to the manufacturer's instruction. The genes associated with TB were selected from the Genomic Wide Association Study using UCSC (http://genome.ucsc.edu/) database. We found that FOXO3 was associated with several diseases including TB (Greer & Brunet, 2008). The candidate SNPs of FOXO3 were selected from the 1,000 Genomes Project data (http://www.internationalgenome.org/) with minor allele frequency (MAF) > 0.02 in the Asian population and MAF > 0.05 in the Chinese Han Beijing population. Finally, four SNPs in FOXO3 were selected in this case–control study. The Agena Bioscience Assay Design Suite V2.0 software (https://agenacx.com/online–tools/) was performed to analyze the amplification and extension primers. The MassARRAY Nanodispenser and MassARRAY iPLEX platform (both from Agena Bios 95% CIence, San Diego, CA, USA) were used to genotype the SNPs. Data management was performed using Agena Bioscience TYPER version 4.0 software as previously described (Zhu et al., 2017).

The patient’s blood was collected in EDTA anticoagulation tubes, and then DNA was extracted using a Blood Genomic DNA Mini Kit (Qiagen, Germany). Quality control of the extracted DNA was performed on a Nanodrop 2000 platform (Thermo, USA), and a Qubit 2.0 fluorometer (Thermo, USA) was used to measure the concentration of DNA. Exome capture and library establishment were performed using Agilent SureSelect Human All Exon V6 (Agilent, USA) following the manufacturer’s protocol. The library was then analyzed by an Agilent Bioanalyzer (Agilent, USA) and loaded on an Illumina HiSeq 2500 (Illumina, USA) for sequencing.
PCR was used to amplify the related exonic regions and intronic regions nearby. Sanger sequencing was then performed to validate the mutations in the proband, sister and parents.
The primers used in PCR are as follows: SARS2_E7-E8 (c.680G > A) F: 5′-CTGT CTCTGGAAGCTTCTATCTGG-3′, SARS2_E7-E8 (c.680G > A) R: 5′-TGACTAGGG CAGAGCAGAGATC-3′, SARS2_E14-E17 (c.1205G > A) F: 5′-CTGCCTTCTCTT CCATCGTTTCC-3′, SARS2_E14-E17 (c.1205G > A) R: 5′-AGTGACTGACTCT GAGGCAGCAA-3′.