GSM9135723: RA2.12w; Mus musculus; RNA-Seq
Mice were euthanized by intraperitoneal injection of pentobarbital sodium (150 mg/kg; diluted 1:2 with 0.9% NaCl physiological) and analyzed ~16 h after the last CS exposure. After perfusion of the heart with PBS, for whole lung analysis, lungs were directly collected into 1 mL PBS (2% FBS). Lungs were processed using the mouse lung dissociation kit (#130-095-927, Miltenyi Biotec) according to the manufacturer's instructions (program 37C_m_LDK_1). After termination of the program, C tubes (#130-093-237, Miltenyi Biotec) were detached from the gentleMACS dissociator and centrifuged shortly at 1500 rpm. Pellets were resuspended in the same supernatant and filtered through a 70 μm cell strainer (#CLS431751, Sigma-Aldrich) into a 50 mL tube. After centrifugation at 1500 rpm for 2 min, the supernatant was discarded and the pellets were resuspended with 1 mL ACK lysis buffer (#A1049201, Thermo Fisher Scientific) for 3-5 min. Again, the suspension was filtered through a 70 μm cell strainer (#CLS431751, Sigma-Aldrich) and spun at 1500 rpm for 2 min. The supernatant was discarded and cells were resuspended in 0.5-1 mL PBS (2% FBS) and used for subsequent flow cytometry experiments. For the bulk RNA seq experiments, per group, 8 (4 and 12 weeks experiment) or 12 (12+8 weeks experiment) mice were used. Samples from each 2 mice (4 and 12 weeks experiment) or 3 mice (12+8 weeks experiment) were pooled into 1 sample resulting in 4 biological replicates per experiment. Per sample 100.000 AM (live CD45+CD11c+SiglecF+ cells) were FACS-sorted into RLT buffer (#74106, Qiagen) on BD FACS Aria flow cytometers from whole lung digests (CS: based on the presence of the tdTom signal; RA: without tdTom signal separation) and subsequently stored in -80°C. After RNA isolation according to the manufacturer's instructions (#74106, Qiagen), samples were used for bulk RNA seq. The RNA samples were quantified using the Qubit RNA HS assay (#Q32852, Invitrogen). The RNA integrity Number (RIN) scores and the DV200 scores were determined by using the TapeStation 2200. For this, the High Sensitivity RNA screen tape (#5067-5579, Agilent) and the High Sensitivity RNA ScreenTape Sample Buffer (5067-5580, Agilent) were used, following the manufacturers instructions. RNA library preparation was performed using the Illumina stranded total RNA Prep, Ligation with Ribo Zero Plus kit (#20040529, Illumina), according to the manufacturer's protocol. A total of 50 ng RNA was used for all libraries as input material. Prepared libraries were quantified using the Qubit 1x dsDNA HS kit (#Q33231, Invitrogen) and checked for fragment size distribution using the TapeStation 2200 using the D1000 DNA screen tape (# 5067-5582, Agilent) with D1000 Reagents (5067-5583). The quantified libraries were pooled in equimolar amounts and sequenced on the NovaSeq 6000 system with 2 x 50 bp paired-end (PE) reads, targeting 50 million PE reads per library.