Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1

Siying Ye; Chris Cowled; Cheng-Hon Yap; John Stambas

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37°C. Single cell suspensions were obtained by 389 filtration through a 40 μm cell strainer and cells were allowed to attach to a petri dish in a 1:1 mixture 390 of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) 391 containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37°C. The non-adherent 392 cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a 393 discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface 394 of two density gradients was collected, washed in HBSS, and re-suspended in SAGM media 395 supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL 396 streptomycin (Gibco), prior to plating on polyester Transwell inserts (0.4 μm pore; Corning) coated 397 All rights reserved. No reuse allowed without permission.