Ribo zero rrna removal kit h m r

For ribosome profiling experiments, A549 cells were grown in a 15 cm dish in culture medium. Cells were infected with PR8 or PR8ΔNS1 as described [23 (link)]. Control cells were not subject to any media changes, while mock infected cells were washed with phosphate-buffered saline (PBS), rocked in 4 mL of Dulbecco’s Modified Eagle’s medium (DMEM) without any supplements for 1 h, then washed with PBS, followed by the addition of culture medium. Twelve hours post infection, cells were harvested and library preparation proceeded according to the manufacturer’s protocol using the TruSeq Ribo Profile for Mammalian kit (Illumina^®, Carlsbad, CA, USA). A list of additional reagents can be found within the manufacturer’s protocol and were used as described within. Depletion of ribosomal RNA was performed using the Ribo-Zero rRNA Removal Kit (H/M/R) (Illumina^®). Sequencing libraries were pooled together in groups of four per lane and sequenced at the Genome Resource Center at The Rockefeller University using the Illumina HiSeq 2500, 50 cycle SR_Multiplexed. Sequencing data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE101760.