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

QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set 426 (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to 427 ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) 428 RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM 429 Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) 430 RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the 431 presence of the sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along 432 with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of 433 consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) was 434 performed as previously described (5) to measure mRNA expression of IL-6, CXCL10, CCL5, 435 suppressor of cytokine signaling (SOCS) 1 and SOCS3 (Fig. 2) , which is known to be up-regulated 436 following HPAI H5N1 infection of A549 cells (5). RNA samples were stored in 0.1 volumes of 3 M 437 Sodium Acetate (pH7.5 in DEPC-treated water) and 2 volumes of 100% Ethanol and submitted to 438 Macrogen Inc. (Seoul, Republic of Korea) for HiSeq analysis. 439