title: NextSeq 500 sequencing; Ribosome profiling and RNA sequencing of Infectious Bronchitis Virus
design: Ribosome profiling and RNA sequencing of Infectious Bronchitis Virus
library_construction_protocol: Primary chick kidney (CK) cells were produced from 2–3 week-old specific pathogen free (SPF) Rhode Island Red chickens. CK cells (0.8 x 10-6 cells/ml) were plated in 10-cm dishes and, upon reaching 100% confluence (two days post-seeding), were washed once with PBS and infected with 9.6 x 10-6 PFU rIBV Beau-R. After 1 hour incubation at 37 °C, 5% CO2, the inoculum was removed and replaced with 10 ml fresh 1x BES (1X minimal essential Eagle's medium [MEM], 0.3% tryptose phosphate broth, 0.2% bovine serum albumin, 20 mM N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 0.21% sodium bicarbonate, 2 mM L-glutamine, 250 U/ml nystatin, 100 U/ml penicillin, and 100 U/ml streptomycin). Cells were harvested at 24 hours post-infection when clear regions of cytopathic effect (CPE) were visible.  Cycloheximide (CHX; Sigma-Aldrich) was added directly to the growth medium (to 100 µg/ml) and the cells incubated for 2 min at 37 °C before rinsing with 5 ml of ice-cold PBS containing CHX (100 µg/ml). Subsequently, dishes were incubated on ice and 400 µl of lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 µg/ml cycloheximide and 25 U/ml TURBO™ DNase (Life Technologies)] dripped onto the cells. The cells were scraped extensively to ensure lysis, collected and triturated with a 26-G needle ten times. Lysates were clarified by centrifugation for 20 min at 13,000 g at 4 °C, the supernatants recovered and stored at −80 °C. Cell lysates were subjected to RiboSeq and RNASeq. The methodologies employed were based on the original protocols of Ingolia and colleagues (Ingolia et al., 2009; 2012), except ribosomal RNA contamination was removed using a commercial RiboZero Gold magnetic kit (Illumina) and library amplicons were constructed using a small RNA cloning strategy (Guo et al., 2010) adapted to Illumina smallRNA v2 to allow multiplexing. The methods used were as described by Chung et al. (2015), except the 5′ and 3′ adapters included seven consecutive randomised bases at the 3′ and 5′ ends (respectively). This facilitated removal of reads duplicated during polymerase chain reaction (PCR) amplification of cDNA libraries (Aird et al; 2011) and reduced ligation bias.
attributes.tag: Experimental Factor: infect
attributes.value: none
attributes.tag: Experimental Factor: protocol
attributes.value: RNASeq