title: GSM4291607: SP-A2(1A0)-F-6hr-1; Mus musculus; RNA-Seq
library_construction_protocol: AM cells were isoalated from Broncho alveolar lavage, QuantSeq 3' mRNA-Seq Library Prep Kit FWD from Illumina (Lexogen, Vienna, Austria) was used to generate mRNA-Seq libraries as per manufacturer's recommendation, followed by deep sequencing on an Illumina HiSeq-2500 as per the manufacturer's instructions. The synthesis of the second cDNA strand is performed by random priming, in a manner that DNA polymerase is efficiently stopped when reaching the next hybridized random primer, so only the fragment closest to the 3′ end gets captured for later indexed adapter ligation and PCR amplification.  The processed libraries were assessed for fragment size distribution and quantity using a BioAnalyzer High Sensitivity DNA kit (Agilent Technologies).  Pooled libraries were denatured and loaded onto a TruSeq Rapid flow cell on an Illumina HiSeq 2500 (Illumina) and run for 50 cycles using a single-read recipe (TrueSeq SBS kit v3, Illumina) according to the manufacturer's instructions.  Illumina CASAVA pipeline (released version 1.8, Illumina) was used to obtain de-multiplexed sequencing reads (fastq files) passed the default purify filter.  These were further subjected to QuantSeq data analysis pipeline on a Bluebee genomics analysis platform (Bluebee, Cambridge, MA).  The differentially expressed genes (DEG) between males and females were identified by using edgeR test method and TCC v1.14.0 R package.  The DEG are selected based on their fold change and their P-value (P < 0.05) for further analyses.  We chose genes for further analysis based on their P-value (P < 0.05) and their expression levels (≥ 2 fold change) in AM cells from K. pneumoniae infected mice.  The fold differences for the identified genes between males and females were determined by dividing a specific individual male gene value by the corresponding specific female gene value and vice versa for the same gene.
attributes.tag: GEO Accession
attributes.value: GSM4291607