title: RNAseq of lung tissue from mice exposed to conventional and renewable diesel exhaust particle
design: RNAseq of lung tissue from mice exposed to conventional and renewable diesel exhaust particle
library_construction_protocol: Lung tissue was collected 24 hours post exposure, snap-frozen in liquid nitrogen and stored at -80 until RNA extraction.  Particles were suspended in Nanopure Diamond water with 0.1 % Tween80. An identical solution of Nanopure Diamond water with 0.1 % Tween80 served as vehicle control. A PFTE filter free from particles extracted with the same procedure was used as an extraction filter control. All solutions were freshly prepared and instilled within 1 h. Detailed description of the instillation protocol can be found in the reference below. For the downstream analysis the combustion particles should be compared to the extraction control (also containing vehicle) while the CB (Printex90) particles should be compared to the vehicle control since they are not combustion particles.   Bendtsen, K.M., Brostrøm, A., Koivisto, A.J. et al. Airport emission particles: exposure characterization and toxicity following intratracheal instillation in mice. Part Fibre Toxicol 16, 23 (2019). https://doi.org/10.1186/s12989-019-0305-5 Extraction of total RNA from lung tissue was performed using RNeasy Mini kit (Qiagen). One day prior to RNA extraction, tissues were placed in prechilled RNAlater®-ICE Frozen Tissue Transition Solution (Invitrogen) at -20 overnight. At the day of extraction, tissue was transferred to RLT buffer supplemented with B-mercaptoethanol (1 % v/v) and homogenized by a bead-homogenizer (Precellys 24) operated at 6500 rpm (20 seconds x 2). Subsequently, tissue lysate was transferred to QIA Shredder spin column (Qiagen, #74124) and centrifuged before supernatant was added to RNeasy column and total RNA was isolated according to manufacturer's instructions with the inclusion of the purification step with DNase I (Qiagen).  RNA sequencing libraries were prepared using Illumina's standard stranded mRNA library preparation protocol with poly(A) enrichment. 