Total rna prep with ribo zero plus library kit

RNA from infected cells were extracted as described above. Sequencing libraries were prepared using the Illumina Total RNA Prep with Ribo-Zero Plus library kit (Cat# 20040525) according to the manufacturer’s guidelines. Briefly, 100 ng of total RNA was first depleted of the abundant ribosomal RNA present in the samples by rRNA-targeted DNA probe capture followed by enzymatic digestion. Samples were then purified by Beckman Coulter RNAClean XP beads (Cat# A63987). Obtained rRNA-depleted RNA was fragmented, reverse transcribed, converted to dsDNA, end repaired, and A-tailed. The A-tailed DNA fragments were ligated to anchors allowing for PCR amplification with Illumina unique dual indexing primers (Cat# 20040553). Libraries were pooled in equimolar concentrations and sequenced on Illumina NextSeq 500 and NextSeq 550 sequencers using high-output cartridges (Cat# 20024907), generating single 150-nt-long reads.

To induce shRNA expression A549-Ace2 cells and the derived shRNA lines were cultured in doxycycline containing media (1 μg/ml) for > 14 days. 2.5x10⁵ cells each were seeded into a 24-well plate and Cells were infected with 2x10⁴ PFU/well (MOI = 0.1) of SARS-CoV-2 (hCoV-19/England/02/2020). At 24 hpi, cells were detached and lysed in Trizol LS. Total RNA extraction was performed following manufacturers recommendation. qRT-PCR was performed using Luna (NEB # E3005L) with gene specific primers (Table S8). RNA sequencing libraries were prepared using the Illumina Total RNA Prep with Ribo-Zero Plus library kit (Cat# 20040525) according to manufacturer’s guidelines. Briefly, 100ng of total RNA was first depleted of the abundant ribosomal RNA present in the samples by rRNA targeted DNA probe capture followed by enzymatic digestion. Samples were then purified by Beckman Coulter RNAClean XP beads (Cat #A63987). Obtained rRNA-depleted RNA was fragmented, reverse transcribed, converted to dsDNA, end repaired and A-tailed. The A-tailed DNA fragments were ligated to anchors allowing for PCR amplification with Illumina dual indexing primers (Cat#20040553). Libraries were pooled in equimolar concentrations and sequenced on an Illumina NextSeq 500 sequencer using a high-output cartridge (Cat# 20024907), generating single 150bp long reads.