GSM6364231: CD,ileum,SARS-CoV-2,patient7_1; Homo sapiens; RNA-Seq
After centrifugation at 2 000 g for 5 minutes, the supernatant was split into two tubes and frozen at -80 °C. 200 µl of TRIzol (Thermo Fisher Scientific) was added to each monolayer well and replicates were collected separately. Cell lysates in TRIzol were vortexed and frozen at -20 °C. Total RNA was isolated according to manufacturer's protocol. Aliquots were taken from samples originating from different monolayer plates to test for the presence of SARS-CoV-2 RNA. After initial quality control using Agilent's Bioanalyzer sequencing libraries were prepared from 25-250ng of total RNA per sample following Roche's strand specific “KAPA RNA Hyper Prep Kit + RiboErase (HMR)” library preparation protocol for double indexed Illumina libraries:   First, the rRNA fraction was depleted using complementary DNA oligonucleotides. rRNA-DNA hybrids and DNA baits were removed by treatment with RNase H and DNase.  RNA was heat-fragmented and subjected to first strand synthesis using random priming. The second strand was synthesized incorporating dUTP instead of dTTP to preserve strand information. After A-tailing Illumina sequencing compatible adapters carrying unique dual indices were ligated (NEXTFLEX® Unique Dual Index Barcodes).  Following bead-based clean-up steps the libraries were amplified using 13 cycles of PCR. Library quality and size was checked with qBit, Agilent Bioanalyzer and qPCR. Sequencing was carried out on an Illumina NovaSeq6000 system in PE100bp mode yielding between 20-40 million fragments per sample.