Effect of Aeroplysinin-1 on gene expression in primary human lung endothelial cells

Word document (RNASeq analysis documentation) describes methods of processing reads.Lysates were prepared by washing cells twice with PBS+, lysed in recommended volume of RLT Lysis buffer from the Qiagen RNeasy RNA Isolation Kit (Cat. No. / ID: 74104) and collected by scraping with a sterile cell scraper. Lysates were vortexed for 20 seconds, centrifuged at max speed for 3 minutes at room temperature at stored at -80C for up to two weeks prior to extraction according to manufacturer’s instructions. Samples had an Agilent Bioanalyzer RNA integrity number above 7 and an OD260/230 ratio of 2.0-2.2.

Library prep was performed using the NEB Ultra II Directional mRNA kit. Sequencing was performed with the Illumina Hiseq2500 platform with high throughput flowcell PE2x125bp (paired end 2x125 base pair configuration), with 25-30 million reads per sample.Reference-based count-based differential gene expression with STAR+HTseq+edgeR, comparing the following 9 sets of samples for HPMECs; (1) DMSO Flu vs Ap Flu, (2) DMSO Flu Vs Ap1 Flu, (3) Ap Flu vs Ap1 flu, (4) DMSO no flu vs Ap no flu, (5)DMSO No flu vs Ap1 no flu, (6) Drug no flu vs Ap1 no flu, (7) DMSO no flu vs DMSO Flu, (8) Ap no flu vs Ap flu, (9) Ap 1 no flu vs Ap1 flu.

The genome reference sequence was GRCh37 primary assembly, Gencode v.28lift37 gene models, downloaded from gencodegenes.org/human/release_28lift37.html. The following small RNA species, which should be excluded by size from the RNA-Seq libraries, are removed from the annotation file to avoid spurious results: Mt_tRNA, miRNA, scRNA, snRNA, snoRNA, vaultRNA.