RNA was extracted from murine lung tissue using the Trizol/Chloroform method. First, about 15 mg of lung tissue was homogenized in TriZol LS reagent (Thermo Fisher Scientific, Waltham, MA, US) using the SpeedMill Plus. The tissue-free supernatant of the homogenate was transferred into a new tube after centrifugation. 50 µl of chloroform was added to 250 µl TriZol Reagent, incubated for 3 minutes at RT, and centrifuged at 12000xg for 15 minutes at 4°C. The aqueous phase was mixed with isopropanol in a 1:2 ratio and incubated at RT for 10 minutes before centrifugation at 12000xg for 10 minutes at 4°C. Afterward, the RNA pellet was washed twice with 75% ethanol. Finally, ethanol was removed, and the pellet was air-dried for 5-10 minutes. The dry pellet was then dissolved in sterile distilled water, and RNA concentration was determined by an ND-1000 NanoDrop spectrophotometer (PEQLAB Biotechnologie GmbH, Erlangen, Germany). Before sequencing, RNA integrity was measured using a 5400 Fragment Analyzer (Agilent Technologies, Santa Clara, CA, US)) RNA concentration and RNA integrity of the samples can be found in Supplemental Table 1.
Library construction and mRNA sequencing were performed by Novogene Co., LTD. (Beijing, China) using the Illumina platform Novaseq 6000 S4 flowcell V1.0, based on the sequencing by synthesis (SBS) mechanism and PE150 strategy. For bioinformatics analysis, raw reads in FASTQ format were first processed using fastp. Reads containing adapters and poly N sequences were removed as well as low quality reads to generate clean data. Clean reads were then mapped to the reference genome (Mus Musculus (GRCm39/mm39)) with the HISAT2 software. Gene expression levels were quantified using the FPKM method. DESeq2 software as well as negative binomial distribution was used for analysis of differential gene expression. For FDR (False discovery rate) calculation the Benjamini-Hochberg procedure was used. A summary of the quality of the sequencing data of each sample can be found in Supplemental Table 2.
Heatmaps of the differentially regulated genes (DEG) were constructed using R (v4.2.2; R Foundation for Statistical Computing, https://www.r-project.org/). Only significant DEGs (p-value < 0.05) were displayed in the heatmaps. Non-significant genes were set to zero and are shown in white. All DEGs used for construction of the respective heatmaps are summarized in Supplemental Table 3a-i.
Library construction and mRNA sequencing were performed by Novogene Co., LTD. (Beijing, China) using the Illumina platform Novaseq 6000 S4 flowcell V1.0, based on the sequencing by synthesis (SBS) mechanism and PE150 strategy. For bioinformatics analysis, raw reads in FASTQ format were first processed using fastp. Reads containing adapters and poly N sequences were removed as well as low quality reads to generate clean data. Clean reads were then mapped to the reference genome (Mus Musculus (GRCm39/mm39)) with the HISAT2 software. Gene expression levels were quantified using the FPKM method. DESeq2 software as well as negative binomial distribution was used for analysis of differential gene expression. For FDR (False discovery rate) calculation the Benjamini-Hochberg procedure was used. A summary of the quality of the sequencing data of each sample can be found in Supplemental Table 2.
Heatmaps of the differentially regulated genes (DEG) were constructed using R (v4.2.2; R Foundation for Statistical Computing, https://www.r-project.org/). Only significant DEGs (p-value < 0.05) were displayed in the heatmaps. Non-significant genes were set to zero and are shown in white. All DEGs used for construction of the respective heatmaps are summarized in Supplemental Table 3a-i.
