Oligo dt

TRIzol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from differently treated cells according to standard instructions. The quality and quantity of RNA in the samples were determined by the uitro-spec2000 mass spectrophotometer. One microgram of RNA was reverse transcribed into cDNA using M-ML-V reverse transcriptase (Promega, Madison, WI, USA), Oligo-dT (TaKaRa, Tokyo, Japan) and random primers (TaKaRa). Next, cDNA was used as a template for qPCR detection using Go Taq qPCR Master Mix (Promega, Madison, WI, USA) and SYBR Green I. The reverse transcription quantitative polymerase chain reaction (qPCR) reaction was performed using the CFX96™ real-time system (Bio-Rad, Hercules, CA, USA) under specific cycling conditions: (1) one cycle at 95 °C for 2 min, (2) 40 cycles at 95 °C for 15 s, and (3) 60 °C for 40 s. Primers for RT-qPCR were designed using the OLIGO 7 primer analysis software (Molecular Biology Insights Inc., Cascade, CO, USA) (Table 1). The relative transcription levels of all genes were calculated by 2^−ΔΔCt and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as the standard. Each data point represents results from three independent experiments.

For the qPCR, total RNA was extracted from the cells using TRIzol (Thermo Fisher), and 5 μg of RNA were used to synthesize cDNA with ReverTra Ace® (Toyobo) and oligo-dT (Takara) according to the manufacturers’ instructions. The transcript levels of the genes were determined using SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara) and a CFX-96 Real-Time system (Bio-Rad). Primers were listed in Supplementary Table S5.

Total RNA was prepared with TRIzol. For quantitative PCR, cDNAs were synthesized with ReverTra Ace (Toyobo) and oligo-dT (Takara) and then analyzed by qRT-PCR with Premix Ex Taq (Takara). For RNA-seq, TruSeq RNA Sample Prep Kit (RS-122-2001; Illumina) was used for library construction, and the sequencing was performed using a NextSeq 500 High Output Kit v2 (75 cycles) (FC-404-1005; Illumina), according to the manufacturer’s instructions. RNA-seq was processed as previously described (47 (link), 48 (link)); briefly, reads were aligned to a transcriptome index generated from the Ensembl annotations, using RSEM (49 (link)), Bowtie2 (50 (link)), and normalized using EDASeq (51 (link)). RNA-seq data are expressed in units of GC-normalized tag counts. DEGs were analyzed by DESeq2 (52 (link)).

Total RNA was isolated using TRIzol reagent (Invitrogen, USA). The first-strand cDNA was synthesized from 1μg of total RNA using Oligo(dT) and random 6 mers oligos for priming (Takara, Dalian, China). The real-time PCR was carried out using the SYBR Premix Ex Taq (Takara, Dalian, China) according to the manufacturer’s protocol with StepOne Plus system (Applied Biosystems, Foster City, CA). The real-time PCR reactions were performed in triplicate and β-actin was used as the internal control. The relative level of target RNAs was evaluated by the comparative CT method 2^−ΔΔCT method. The primer sequences of each gene are shown in Supplementary Table 1.

The gene expression levels in the lung and trachea were quantified using quantitative real-time PCR. The total RNA was isolated with Trizol (Invitrogen, San Diego, CA, USA). The quality and quantity of the RNA were determined by agarose gel electrophoresis and a biophotometer (Eppendorf, Germany), respectively. RT reactions (10 μL) contained 500 ng total RNA, 5 mmol/L MgCl₂, 1 μL of RT buffer, 1 mmol/L dNTP, 2.5 U AMV, 0.7 nmol/L oligo d (T) and 10 U ribonuclease inhibitor (TaKaRa, Dalian, China). Real-time PCR analysis was conducted using the Applied Biosystems 7500 Real-time PCR System (Applied Biosystems, Foster, CA, USA). Each RT reaction served as a template in a 20 μL PCR reaction that contained 0.2 μmol/L of each primer and SYBR green master mix (Takara, Dalian, China). The real-time PCR reactions consisted of pre-denaturation at 95°C for 10 seconds followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing and extension at 60°C for 40 seconds. A standard curve was plotted to calculate the efficiency of the real-time PCR primers. β-actin was used as the housekeeping gene, and the results of the relative mRNA quantification were verified with the β-actin levels (ΔCT). The comparative CT method (2^−ΔΔCT) was used to quantitate the mRNA expression in accordance with Wang et al [38 (link)]. The primer sequences are listed in Table 2.