title: DEP1_69
description: Protocols: 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.
attributes.tag: strain
attributes.value: C57BL/6Tac
attributes.tag: individual
attributes.value: intratracheal instillation exposure, euthanasia and collection of lung tissue 24 hours post exposure
attributes.tag: organism
attributes.value: Mus musculus
attributes.tag: organism
attributes.value: Mus musculus
attributes.tag: collection date
attributes.value: not collected
attributes.tag: sex
attributes.value: female
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: common name
attributes.value: house mouse
attributes.tag: organism part
attributes.value: lung
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: age
attributes.value: 8 weeks
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected