Reverse transcriptase kit

Total RNA was extracted from indicated immunocytes using TRIzol (Invitrogen) and cDNA was further synthesized using Reverse Transcriptase Kit (Qiagen). Quantitative real-time PCR was performed using the Power Up SYBR Green Master Mix (ABI) to quantify the expression levels of genes (actin as the internal control). PCR with real-time fluorescence detection was performed on ABI StepOnePlus^TM (Applied Biosystems). Data analysis was performed using ABI Stepone software (Applied Biosystem). The specific primers were designed according to their corresponding CDS regions and listed in Supplementary Materials.

Total mRNA was purified from peripheral blood and induced sputum cells using Trizol (Invitrogen) and quantified by an ultraviolet spectrophotometer (Thermo Fisher Scientific, USA) [36 (link)]. 1 µg RNA was reversely transcribed into cDNA using Reverse Transcriptase Kit (Qiagen, Netherlands) in accordance to the manufacturer’s instructions [37 (link)]. Then, quantitative RT-PCR was performed using SYBR® Premix Ex Taq™ II system (TaKaRa, Japan) with the CFX96 Touch™ Real-Time PCR Detection System (Bio‐Rad, USA). The PCR conditions were as follows: 95 ℃ for 30 s, 40 cycles of 95 ℃ for 15 s, and 60 ℃ for 30 s. Resulting mRNA levels were normalized to β-actin and expressed as a fold change relative to control samples. The sequences of the primers used are as follows: FOXO3 (forward, CGG ACA AAC GGC TCA CTC T; reverse, GGA CCC GCA TGA ATC GAC TAT), TP53 (forward, AAG TCT GTG ACT TGC ACG TAC TCC; reverse, GTC ATG TGC TGT GAC TGC TTG TAG) and β-actin (forward, TTC CAG CCT TCC TTC CTG GG; reverse, TTG CGC TCA GGA GGA GCA AT).

Total RNA was harvested with Trizol reagent. 1 µg of total RNA was reverse transcribed into cDNA using the reverse transcriptase kit (Qiagen company, Frankfurt, Federal Republic of Germany). The primers used to amplification of Nrf2 and GAPDH were listed Table 1. The water was served as negative control.Table 1

Sequences of primers used for amplification of target genes

Gene	Primer nucleotide sequence
Nrf2	Forward: 5′-CACATCCAGTCAGAAACCAGTGG-3′
	Reverse: 5′-GGAATGTCTGCGCCAAAAGCTG-3′
GAPDH	Forward: 5′-AACGGATTTGGTCGTATTG-3′
	Reverse: 5′-GGAAGATGGTGATGGGATT-3′

Total RNA was extracted from ILNs cells using an RNeasyPlus Mini Kit (Qiagen, Valencia, CA, USA), followed by reverse transcription using the Reverse Transcriptase Kit (Qiagen, Valencia, CA, USA). Primers used for real-time PCR assays were: AID—F: GCCACCTTCGCAACAAGTCT/R: CCGGGCACAGTCATAGCAC; Bcl-6: F: CACACCCGTCCATCATTGAA/R: TGTCCTCACGGTGCCTTTTT; BLIMP-1: F: GGCTCCACTACCCTTATCCTG/R: TCCTTTTGGAGGGATTGGAGTC; IL-21—F: TGAAAGCCTGTGGAAGTGCAAACC/R: AGCAGATTCATCACAGGACACCCA; and GAPDH: F: TTCACCACCATGGAGAAGGC/R: GGCATGGACTGTGGTCATGA.
GAPDH levels were used to normalize the mRNA. Real-time quantitative PCR was performed using an automated sequencer (Model 7500; Applied Biosystems, Carlsbad, CA, USA) with specific primers and SYBR Green (Applied Biosystems, Carlsbad, CA, USA). The amplification results were analyzed using Sequence Detection System (SDS) software (Applied Biosystems, Carlsbad, CA, USA), and a normalized expression was calculated [18 (link)].

Total RNA was extracted from mice lungs using Trizol reagent and converted (2 μg RNA) to cDNA using Qiagen reverse transcriptase kit according to manufacturer’s instruction. Specific primers for genes GATA 3^{81 (link)} and GAPDH^{82 (link)} were used to amplify using SYBR Premix Ex Taq master mix in ABI 7500 (Table 2). After normalizing the GAPDH mRNA level, the data were evaluated using the Ct (double delta Ct) method and displayed as fold change.Table 2

List of primers.

Sr. no.	Gene	Sequence	Base pairs
1.	GATA-3 F	5′-AGGGACATCCTGCGCGAACTGT-3′	22
2.	GATA-3 R	5′-CATCTTCCGGTTTCGGGTCTGG-3′	22
3.	GAPDH F	5′CTCATGACCACAGTCCATGC′3	20
4.	GAPDH R	5′CACATTGGGGGTAGGAACAC′3	20
ChIP specific primer sequence
5.	BCL2 F	5′-GTGGATGACTGAGTACCT-3′	18
6.	BCL2 R	5′-CCAGGAGAA ATCAAACAGAG-3′	20
7.	CCL2 F	5'-ATGTGAGAGCGCCACTCTTT-3'	20
8.	CCL2 R	5'-TGGTAGCTCTCTGCCCTGTT-3	20

The sequences of TTCC isoforms, Pgp, and β-actin mRNAs were acquired from the National Centre for Biotechnology Information. Primers (sequences listed in Table 1) were synthesized using Primer-Basic Local Alignment Search Tool online software and ordered from Imperial Life Sciences Pvt. Ltd. (Gurugram, Haryana, India).
Cells were grown in duplicate in 6-well plates, and total RNA was extracted manually using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). Next, 1 μg RNA from each group was used to prepare cDNA using the Reverse Transcriptase kit (Qiagen, Hilden, Germany). Real-time PCR was performed using SYBR Green Master Mix (Bio-Rad Laboratories Inc., Hercules, CA) in a real-time PCR instrument (Bio-Rad Laboratories Inc., Hercules, CA). Thermal cycling conditions were as follows: initial denaturation at 95 °C for 2 min followed by 40 cycles of denaturation at 94 °C for 10 s, annealing at 55 °C for 30 s, and extension at 68 °C for 1 min/kb. The final extension was at 68 °C for 10 min. The negative control of each gene was included in the reaction mixture by omitting cDNA samples. mRNA expressions were normalized with β-actin expression. The relative expression of each test gene was measured against the Ca_v3.1 mRNA expression of the negative control group.