GSM9277360: 62_T190; Homo sapiens; RNA-Seq
Bronchial epithelial cells (BECs) from children with asthma (n=37) and healthy children (n=3)  ages 6-18 years of age were obtained from subjects while under general anesthesia for an elective surgery using 4-mm Harrell unsheathed bronchoscope cytology brushes (CONMED®) inserted through an endotracheal tube as we have previously described (8, 42). Cells were then seeded onto T-25 cell culture flasks (Corning®) precoated with type I collagen and proliferated under submerged culture conditions using PneumaCult™EX-Plus medium (Stemcell™) (8). BECs (passage 2 or 3) were proliferated on transwells under submerged culture conditions using PneumaCult™EX-Plus medium (Stemcell™) for 4-7 days then differentiated to an organotypic pseudostratified ciliated state (Supplemental Figure 1) for 21 days at ALI using PneumaCult™ALI medium (Stemcell™) as previously described (8, 43–45). We have previously demonstrated stability of gene expression in primary BEC ALI cultures between passages 1 and 3 (46).RNA was collected from BEC cultures pre-infection and at 2d, 4d, 7d, and 10d post RV infection. To collect RNA from BEC cultures, media was first removed from the basolateral chamber of transwells. 800 µL of lysis buffer (Invitrogen®, Product No. AM1912) was added to the apical surface of cultures. A pipet tip was then used to gently scratch each apical well in a crosshatch pattern to loosen BECs from the transwell membrane. RNA was extracted using the RNAqueous™ kit for total RNA isolation (Thermo Fisher Scientific). BEC cultures from SE (n=6) and NSE (n=6) donors were treated with IFN-β or CXCL10 starting 2 days prior to RV infection and continued for 2 days following RV infection. Recombinant human recombinant human IFN-β (1 ng/mL; AbcamⓇ) or CXCL10 (2 ng/mL; R&D SystemsⓇ) was added to BEC ALI medium starting 2 days prior to RV infection and refreshed with each medium change (every other day). Two days following initiation of treatment with IFN-β or CXCL10 BEC cultures were infected with human RV-A16 at an MOI of 0.5 as described above. RNA was collected from BEC cultures pre-infection and 2d following RV infection. Total RNA was used to construct libraries by using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara, San Jose, Calif), with reverse transcription followed by PCR amplification to generate full-length amplified cDNA. Sequencing libraries were constructed by using the NexteraXT DNA sample preparation kit with unique dual indexes (Illumina, San Diego, Calif) to generate Illumina-compatible barcoded libraries. Libraries were pooled and quantified using a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, Mass). Sequencing of pooled libraries was carried out on a NextSeq 2000 sequencer (Illumina) with paired-end 53-base reads with a target depth of 5 million reads per sample