Qubit high sensitivity assay

RNA was extracted using the BioRobot EZ1 and EZ1 RNA Universal Tissue kit (Qiagen) and DNase-treated with Ambion DNA-free DNA removal kit (Invitrogen, USA) according to the manufacturer’s protocols. RNA quantity and quality were assessed using NanoDrop ND-1000 Spectrophotometer (Nanodrop Technologies) and Agilent 2100 Bioanalyzer (RNA 6000 Nano LabChip kit, Agilent Technologies).
DNA isolation was performed using the DNeasy Blood & Tissue Kit (Qiagen, Cat. No. #69506) according to the manufacturer’s protocol. Liver samples were pre-treated with RNase A (provided in the Qiagen kit, 50 ng/µL, 10 min at room temperature) immediately followed by proteinase K treatment (New England Biolabs, #8102S 20 µg/µL, 1.5 h at 55°C). DNA was eluted in Milli Q water, and its quantification was performed using the Qubit High Sensitivity Assay (Life Technologies #Q32854).

NEB Next Multiplex Small RNA Library Prep Kit was utilized to prepare the libraries. First, 200 ng of total RNA was denatured under elevated temperatures. The denatured RNA was then ligated to a 3ʹ SR adapter, followed by SR-RT primer annealing to convert the free single-stranded adapter to the double-stranded DNA molecule. Reverse transcriptase was used to copy the 3ʹ SR adapter-ligated molecules into first-strand cDNA. Following that, the adapter-ligated products were purified and enriched using the thermal conditions provided here: initial denaturation at 94 °C for 30 s; 15 cycles at 94 °C for 15 s, 62 °C for 30 s, 70 °C for 15 s; final extension at 70 °C for 5 min. After that, PCR products were purified, and fragment size distribution was examined on Tape Station using D1000 DNA Screen Tapes (Agilent, Cat# 5067–5582), accompanied by size selection on 4% E-gels. The Qubit High Sensitivity Assay (Invitrogen, Cat# Q32852) was used to quantify the libraries that had been produced. Before cluster amplification on the Illumina flow cell, the resulting libraries were merged and reduced to the final optimum loading percentage. The cluster flow cell is then put into the HiSeq 2500 instrument to generate 25 M 50 bp single-end reads.

Genomic DNA (gDNA) was extracted from second passage of MTB grown on LJ slants using CTAB method [32 (link)]. DNA (µg/ml) was quantitated using Qubit High sensitivity assay (Invitrogen). 0.5 ng/µl of input gDNA was used for library preparation, that was carried out using Nextera XT DNA library kit (15,032,355, 15,052,163). Prepared libraries were then sequenced using the Illumina MiSeq platform (Illumina). Sequence was performed on pair end reads of 2 × 250 bp using the MiSeq reagent kit v2 [33 ]. Furthermore, 10 of the BDQ R/I pre-XDR/XDR MTB isolates that had low average genome coverage (< 10x) were re-sequenced using the Illumina MiniSeq platform (Illumina). MiniSeq high output reagent cartridge (15,073,286) was used.

For bulk RNA sequencing, total RNA was isolated from stimulated organoids using RNeasy Micro Kit (Qiagen) and quantified using Qubit High Sensitivity Assay (Invitrogen). For scRNA-seq, stimulated organoids from eight wells were pooled and dissociated into single cells, bigger fragments removed by a 30-µm strainer, and 6000 cells were processed using the ChromiumNext GEMSingle Cell 3ʹReagent Kits Dual Index Kit (10x Genomics). The concentration of the prepared library was analyzed, and fragment size confirmed using High Sensitivity DNA Kit (Agilent).
The process of bulk RNA sequencing was carried out at Novogene using an Illumina Novaseq HiSeq Pair-Ended 150 bp, with a sequencing depth of 9 G, equivalent to 30 million reads for each sample. Similarly, scRNA-seq was executed, achieving a sequencing depth of 90 G for every sample.

The samples from TB-infected individuals from both India and Brazil sites were collected at baseline, 2, and 6 months of treatment. Samples for the HC and DM groups were collected at baseline. Whole blood (5 mL) was collected PAXgene Blood RNA tubes (Qiagen, catalog #762165) and frozen at − 80 °C. RNA was extracted using the PAXgene Blood RNA kit (Qiagen, catalog #762174) and quantified using Qubit RNA assay HS (Invitrogen, Cat #Q32852). RNA purity was checked using QIAxpert, and RNA integrity was assessed on TapeStation using RNA HS ScreenTapes (Agilent, Cat #5067-5579). NEB Ultra II Directional RNA-Seq Library Prep kit protocol was used to prepare libraries for total RNA sequencing. Prepared libraries were quantified using Qubit High Sensitivity Assay (Invitrogen, Cat #Q32852), pooled and diluted to final optimal loading concentration before cluster amplification on Illumina flow cell. Once the cluster generation was completed, the cluster flow cell was loaded on Illumina HiSeq 2500 instrument to generate paired end reads at MedGenome in Bangalore, India.

Libraries for 768 SARS-CoV-2-positive RNA samples were prepared for sequencing following the Illumina Covidseq workflow (Illumina Inc., San Diego, CA USA) in accordance with the manufacturer’s specifications. The extracted RNA was reverse-transcribed to generate cDNA using random hexamers. Generated cDNA was amplified by dividing the cDNA into two pools according to the designed primers to produce 98 amplicons spanning the SARS-CoV-2 genome along with 11 control amplicons for human RNA. The two pools were then recombined to be simultaneously fragmented and tagmented. The tagmented amplicons were subjected to a post-tagmentation clean-up step and amplified once more with the addition of indexes to each sample using IDT for Illumina Nextera Unique Dual Indexes set A, B C and D (384 indexes for 384 samples). The indexed libraries were then pooled and cleaned in batches of 96 and quantified using the Qubit High-Sensitivity Assay on a Qubit 4.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Each pool was then diluted to a concentration of 4 nM, and 25 μL from each pool was transferred into a new microcentrifuge tube to be denatured and then diluted as specified by the manufacturer of the NovaSeq 6000 SP flow cell workflow (Illumina Inc., San Diego, USA). The libraries were then loaded into the Illumina NovaSeq 6000 system for dual-indexed paired-end sequencing.

Prepared libraries were quantified using Qubit High Sensitivity Assay (Invitrogen, Cat# Q32852). The obtained libraries were pooled and diluted to a final optimal loading concentration before cluster amplification on an Illumina flow cell. Once the cluster generation is completed, the cluster flow cell is loaded into an Illumina HiSeq 4000 instrument to generate 60 M, 100 bp paired-end reads. More than 2000 dysregulated genes were selected and classified into two groups: upregulated and downregulated genes. This list of genes was filtered twice, keeping only the significant genes with a p value of <0.05 and a fold change of greater than 1.8.
These different genes were then classified into pathways by using Panther version 16.0, and the gene panel selected for our study was based on the pathways involved in chronic inflammation and carcinogenesis with the most significant fold change.