RNA-seq of a H1N1 infected airway organoid from cow tissue, 1
RNA was isolated using Monarch Total RNA Miniprep Kit (New England BioLabs). Total RNA concentration was calculated by Quant-IT RiboGreen (Invitrogen). To assess the integrity of the total RNA, samples were run on the TapeStation RNA screentape (Agilent). Only high-quality RNA preparations, with RIN greater than 7.0, were used for RNA library construction. Each library was independently prepared with 10 ng of total RNA from each sample by SMARTer ultra-low input RNA Sample Prep Kit (Clontech Laboratories) following the manufacturers protocol. In brief, polyA containing mRNA molecules were purified using polyTattached magnetic beads and the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using SMARTerscribe reverse transcriptase (Clontech Laboratories) and 3 SMART CDS primers. Second strand cDNA synthesis was performed using DNA Polymerase I, RNase H and dUTP. These cDNA fragments then went through an end repair process, the addition of a single A base, and then ligation of the Illumina adapters. The products were then purified and enriched with PCR to create the final cDNA library. The libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms according to the qPCR Quantification Protocol Guide (KAPA BIOSYSTEMS) and qualified using the TapeStation D1000 ScreenTape (Agilent). Indexed libraries were then submitted to Illumina NovaSeq, and the paired-end (2  100 bp) sequencing was performed by Macrogen Incorporated.