GSM9702550: 16; 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 integrity and quantity were assessed using an RNA Biochip and Qubit RNA Fluorescence Dye (Invitrogen). The RNA sample received was quantified using Qubit 4.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and RNA integrity was checked using TapeStation (Agilent Technologies, Palo Alto, CA, USA). The NEBNext Ultra II RNA Library Prep Kit for Illumina & NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA), including clustering and sequencing reagents, was utilized according to the manufacturer's recommendations. Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94°C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3' ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered onto a flowcell on the Illumina NovaSeq instrument according to manufacturer's instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the NovaSeq Control Software (NCS). Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.20 software. One mis-match was allowed for index sequence identification.