GSM9702620: CASP11-2-lung-day-4; Mus musculus; RNA-Seq
Total RNA was extracted from the left lung lobe using TRIzol reagent (Thermo Fisher Scientific, Cat. 15596026) according to the manufacturer's instructions. RNA cleaning and concentration were performed using the Zymo Research RNA Clean & Concentrator-5 kit (Cat. R1015). RNA quantity was assessed using a Qubit 4.0 Fluorometer (Thermo Fisher Scientific) and RNA integrity was assessed using TapeStation (Agilent Technologies). Library preparation was performed at GENEWIZ from Azenta Life Sciences (South Plainfield, NJ, USA) using the NEBNext Ultra II RNA Library Prep Kit for Illumina with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's recommendations. Briefly, mRNAs were enriched using Oligo-d(T) beads, fragmented for 15 minutes at 94°C, and used as template for first- and second-strand cDNA synthesis. cDNA fragments were end-repaired and adenylated at 3' ends, followed by ligation of universal adapters, index addition, and library enrichment by limited-cycle PCR. Library quality and quantity were assessed using the Agilent TapeStation (Agilent Technologies), Qubit 4.0 Fluorometer (Thermo Fisher Scientific), and quantitative PCR (KAPA Biosystems). Sequencing libraries were multiplexed and clustered onto a flowcell on the Illumina NovaSeq instrument and sequenced using a 2×150 bp paired-end (PE) configuration. Image analysis and base calling were conducted by the NovaSeq Control Software (NCS). Raw sequence data (.bcl files) generated from the Illumina NovaSeq instrument were converted into FASTQ files and demultiplexed using Illumina bcl2fastq 2.20 software, allowing one mismatch for index sequence identification.