GSM5608913: MGI0061_1_Young_single-cell; Mus musculus; RNA-Seq
At various time points after infection, lungs were manually excised and immersed in RNAlater solution (Applied Biosystems). Total RNA was isolated from lung homogenates using RNeasy Mini Kits (Qiagen).  Reverse transcription was carried out using the Applied Biosystems High Capacity cDNA Reverse Transcription Kit.  Real Time PCR was performed on the ABI 7500 Fast Real-Time PCR system. RNA from whole lung homogenates were converted to cDNA libraries for sequencing using the RiboZero Kit (Sigma Aldrich).  Libraries generated from BAL RNA were generated using the SMARTer kit (Clontech).  Sequencing was carried out on a NovaSeq6000 instrument (Illumina).  RNA-seq reads were aligned to the Ensembl release 76 assembly with STAR version 2.0.4b.  Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.5.  Transcript counts were produced by Sailfish version 0.6.3.  Sequencing performance was assessed for total number of aligned reads, total number of uniquely aligned reads, genes and transcripts detected, ribosomal fraction known junction saturation and read distribution over known gene models with RSeQC version 2.3. All gene-level and transcript counts were then imported into the R/Bioconductor package EdgeR and TMM normalization size factors were calculated to adjust for samples for differences in library size.  Genes or transcripts not expressed in any sample were excluded from further analysis.  The TMM size factors and the matrix of counts were then imported into R/Bioconductor package Limma and weighted likelihoods based on the observed mean-variance relationship of every gene/transcript were then calculated for all samples with the Voom function (REF).  Performance of the samples was assessed with a spearman correlation matrix and multi-dimensional scaling plots.  Gene/transcript performance was assessed with plots of residual standard deviation of every gene to their average log-count with a robustly fitted trend line of the residuals. Generalized linear models with robust dispersion estimates were then created to test for gene/transcript level differential expression. Differentially expressed genes and transcripts were then filtered for FDR adjusted p-values less than or equal to 0.05.  To examine age-associated differential gene expression in BAL samples (FIG), Log2 normalized Voom adjusted data was analyzed using one-way ANOVA on ranks.