RNA-seq of air-liquid interface co-cultures of A549 and THP-1 cells exposed to Saharan dust or DQ12 against unexposed controls
RNA-seq of air-liquid interface co-cultures of A549 and THP-1 cells exposed to Saharan dust or DQ12 against unexposed controls
After 24 hours of exposure, the transwell membranes with the air-liquid interface co-cultures were rinsed with ice-cold PBS twice, both on the apical and basolateral sides. Subsequently, the membranes were cut out of their frame with a scalpel, immersed into 500 µL ice-cold TRIzol reagent (ThermoFisher, #15596026), vortexed, and frozen at -80°C.  A549 (ATCC, #CCL-185) cells were cultured in RPMI-1640 (containing L-glutamine) (ThermoFisher, #21875) substituted with 10 % FCS (Merck, #F7524) and 1 % P/S (Sigma, #P0781), hereinafter A549 medium. After thawing, A549 cells were used at passages 2–20. THP 1 (ATCC, #TIB-202) cells were cultured in RPMI-1640 (containing L-glutamine and 25 mM HEPES) (ThermoFisher, #52400) substituted with 10 % FCS, 1 % P/S, 1 mM sodium pyruvate (ThermoFisher, #11360070), 0.7 % D-glucose (Sigma, #G8769) and 50 nM 2-mercaptoethanol (ME) (ThermoFisher, #31350010), hereinafter THP 1 medium. After thawing, THP-1 cells were used at passages 5–15. For air-liquid interface (ALI) co-cultures, on the first day, 1.12*105 A549 cells in 0.5 mL A549 medium were seeded on the apical side of transwell filters (Corning, #3460). At the basolateral side, 1.0 mL A549 medium was used. On the second day, the apical and basolateral medium was removed and fresh A549 medium added. In parallel, 3*106 THP-1 cells were differentiated with 100 nM phorbol 12-myristate-13-acetate (PMA) (Sigma, #P1585) in 25 cm² flasks (Greiner, #690175) for 24 h. On the third day, the basolateral medium removed and fresh A549 medium was added and the apical A549 was removed. The differentiated THP-1 cells were detached with accutase (Sigma, #A6964). 4.4*104 differentiated THP-1 cells in 0.5 mL THP-1 medium without ME were seeded on the apical side of the confluent A549 cell layer. After for 2 h, the apical medium was removed. The co-culture was maintained in contact with air for 22–26 h until exposure on the fourth.  For exposure a Vitrocell Cloud 12α system was used. The ALI co-cultures were placed onto 3 mL A549 medium containing 25 mM HEPES (Sigma, #H0887). In the exposure chamber, 3x 500 µL 4 mg/mL Saharan dust or DQ12 suspended in 1.25 % PBS in endotoxin-free H2O was nebulized. In parallel, in the control chamber, 236 µL 1.25 % PBS in endotoxin-free H2O was nebulized. Before each nebulization, the humidity in the chambers was left to saturate for 30 min. After each nebulization, sedimentation was observed via Vitrocell quartz crystal microbalance, and at the end of sedimentation the chambers were lifted. After exposure, the ALI co-cultures were placed on 1.2 mL A549 medium and incubated for 24 h until sample collection.  The transwell membranes with the cells of the co-culture immersed in TRIzol reagent were left to melt at room temperature. The samples were vigorously shaken at room temperature for 10 min using a Thermomixer 5436 (Eppendorf). Subsequently, 100 µL of ice-cold chloroform (Roth, #7331.2) was added, vortexed for 15 s, left at room temperature for 10 min, and centrifuged at 4°C, 12,000 g for 15 min. The upper aqueous phases were transferred to a new vial and 250 µL 2-propanol (Sigma-Aldrich, #I9516) were added. The mixtures were vortexed for 15 s, left at room temperature for 10 min, and centrifuged at 4°C, 12,000 g for 15 min. The supernatants were discarded and 900 µL 75% ethanol (Roth, #9065.4) in nuclease-free H2O (Qiagen, #1039498) was added. The samples were briefly vortexed and centrifuged at 4°C, 12,000 g for 5 min. The supernatants were discarded and the samples were left to dry for about 10 min. The dry RNA pellets were dissolved in 30 µL nuclease-free H2O. The RNA samples were treated with DNase I (Merck, # AMPD1) to digest genomic DNA. An RNA quality check was performed using a High Sensitivity RNA ScreenTape Analysis kit (Agilent, #5067-5579). Only RNA with an RNA integrity number equivalent of ≥ 7 was subjected to library preparation. 50 ng total RNA of each sample was used as input for library preparation using the PCR-cDNA Barcoding kit (Oxford Nanopore Technologies, #SQK-PCB109) according to the manufacturer's instructions (protocol version: PCB_9092_v109_revD_10Oct2019).