title: GSM5508482: NatG12D 2; Mus musculus; RNA-Seq
library_construction_protocol: All steps of tissue extraction, lysis, and RNA extraction were performed in a fume hood with tools sprayed with RNAZap. The lungs were harvested, stored in RNALater overnight, then stored at -80C for long-term storage. RNA extraction was performed with RNEasy Kit using QIAShredder columns for successful lysis and RNase-Free DNAse Set for removal of genomic DNA. Extracted total RNA quality and concentration was assessed on Fragment Analyzer (Agilent Technologies) and Qubit 2.0 (ThermoFisher Scientific), respectively.  Samples with RIN less than 7 were not sequenced. RNA-seq libraries were prepared using the commercially available KAPA Stranded mRNA-Seq Kit (Roche).  In brief, mRNA transcripts were captured from 500 ng of total RNA using magnetic oligo-dT bead.  mRNA was then fragmented using heat and magnesium, and reverse transcribed using random priming.  During second strand synthesis, the cDNA:RNA hybrid was converted into to double-stranded cDNA (dscDNA) and dUTP was incorporated into the second cDNA strand, effectively marking this strand.  Illumina sequencing adapters were ligated to the dscDNA fragments and amplified to produce the final RNA-seq library.  The strand marked with dUTP was not amplified, allowing strand-specificity sequencing.  Libraries were indexed using a dual indexing approach allowing for all the libraries to be pooled and sequenced on the same sequencing run.  Before pooling and sequencing, fragment length distribution for each library was first assessed on a Fragment Analyzer (Agilent Technologies).  Libraries were also quantified using Qubit.  Molarity of each library was calculated based on qubit concentration and average library size.  All libraries were then pooled in equimolar ratio and sequenced.  Sequencing was done on an Illumina NovaSeq 6000 sequencer. The pooled libraries were sequenced on one lane of an S-Prime flow cell at 50bp paired-end.  Sequencing was done on an Illumina NovaSeq 6000 sequencer.  The pooled libraries were sequenced on one lane of an S-Prime flow cell at 50bp paired-end.  Once generated, sequence data was demultiplexed and Fastq files generated using bcl2fastq v2.20.0.422 file converter from Illumina.