title: GSM7908833: 20-LD-OP-Veh-Liv; Mus musculus; RNA-Seq
library_construction_protocol: Lower right lobe of the lung tissues was immediately placed in RNAlater solution, then held on ice for 4–5 hours, and then stored at -80ºC until the RNA extraction procedure. RNA extraction was performed using RNeasy Mini Kits from Qiagen (Valencia, CA). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads.  Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNAs 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 limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on flowcells. After clustering, the flowcells were loaded on to the Illumina HiSeq instrument (4000 or equivalent) 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 HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.