GSM8490315: Alveolus Lung-on-a-chip uninfected, 24 hr, biol rep 1; Homo sapiens; RNA-Seq
ALoCs were removed from the Zoe and Pods. Top and bottom channels were washed 3X with 200 µL of ice-cold 1X DPBS (-Ca+/-Mg+). 200 µL of TRIzol reagent (Invitrogen 15596026) was pipetted in each ALoC by rapidly pressing and releasing the plunger 3X before collecting the supernatant in an RNase-free 1.5 mL tube. This process was repeated once more for a total of ~400 µL of supernatant. TRIzol was added to each tube of RNA to a final volume of 1 mL. The RNA was stored at -80°C before extraction using the Qiagen RNeasy Mini Columns (74106). AT and macrophage lysates were thawed on ice and incubated at room temperature for 5 min. 200 µL of chloroform per 1 mL of lysates was added to each tube, shaken vigorously for 15 sec., incubated at room temperature for 2-3 min, and centrifuged for 5 min at 12,000xg at 4°C. The aqueous phase was transferred to fresh RNase-free tubes and the rest of the RNA extraction was performed according to the Qiagen RNeasy Mini Column Kit. Extracted RNA was stored at -80°C until use for library preparation and RNA-sequencing. Samples were analyzed on an Agilent Tapestation 4200 to determine level of degradation to ensure that only high quality RNA was used (RIN Score 8 or higher). We used a Qubit® 4.0 Fluorimeter (ThermoFisher) to determine RNA concentration prior to starting library prep. One microgram of total DNAse treated RNA was then prepared with the TruSeq Stranded mRNA Library Prep Kit (Illumina). Poly-A RNA was purified and fragmented before strand-specific cDNA synthesis. cDNA was then A-tailed and indexed adapters ligated. After adapter ligation, samples were PCR amplified and purified with AmpureXP beads, then validated again on the Agilent Tapestation 4200. Before being normalized and pooled, samples were quantified by Qubit then sequenced on the Illumina NextSeq 2000 using a P2-100 flowcell.