title: GSM8036810: A549 cells infected with ZIKV sfRNAnull replicate 2 Ribo-seq; Homo sapiens; OTHER
library_construction_protocol: In brief, ribosome protected fragments were collected from duplicate samples of A549 cells infected with ZIKV wild type or sFRNAnull mutant at 20 hpi. A p100 dish of A549 cells was incubated with cycloheximide (0.1 μg/ml) in PBS for 5 minutes. The PBS was removed, and the cells were treated with Lysis buffer (20 mM total Tris HCL pH 8.0; 140 mM KCl; 1.5 mM MgCl2; 100 μg/ml Cycloheximide; 1% Triton; 40 U/ml SUPERas In (Ambion #AM2694); 10 μl/ml Protease Inhibitor Cocktail (Calbiochem #535140-1ML)). The mix was clarified for 10 minutes at 10,000g at 4°C. RNase I (100 Units, Ambion #AM2295) and DNase (10 Units, Ambion #AM2238) were added to the supernatant and incubated at room temperature for 45 minutes with gentle mixing. Digested extracts (50 μl) were then overlaid onto s-40 columns following the manufacturer's instructions. RNA was extracted using Trizol (Invitrogen, #800868). RNA for each sample for RNA-seq was collected in parallel with the Ribosome profile experiment for RNA preparation. RNA was extracted using Trizol (Invitrogen, #800868). Ribosome protected fragments (RPF) were separately excised in a 15% urea gel from 28-to-38 nucleotides. RNA was eluted overnight in 300 mM NaOAc pH 5.5; 1 mM EDTA; 0.1U/μl SUPERas In (Ambion #AM2694), followed by ethanol precipitation. RFP were 3'-dephosphorylated with polynucleotide kinase (10 U, PNK, New England Biolabs) in T4 polynucleotide kinase buffer (without ATP) for 1 h at 37°C, followed by a 10 min incubation at 75°C and ethanol precipitation. All RNA samples were resuspended and ligated to 3' adaptor (30 μM /5rApp/ATCTCGTATGCCGTCTTCTGCTTG/3ddC/) using T4 RNA Ligase 2, truncated K227Q (2 U, New England Biolabs; #M0351S) and RNAseOUT Recombinant Ribonuclease Inhibitor (5 U, Invitrogen, #10777-019) for 6 h at 20°C. Ligation reactions were separately in a 10% urea gel, and fragments from 50-to-65 nucleotides for the RPF and 50-to-75 nucleotides were excised, eluted, and precipitated from the gel. The 3'-ligation products were then 5'-phosphorylated with polynucleotide kinase (15 U, PNK, New England Biolabs) in T4 polynucleotide kinase buffer supplemented with 1 mM ATP for 30 minutes at 37°C followed by RNA precipitation in ethanol. The 5'-ligation was performed using T4 RNA Ligase 1 (ssRNA Ligase, 10 U New England Biolabs, #M0204S) for 6 hrs at 20°C with barcoded 5´-Adaptor (100 μM, GUUCAGAGUUCUACAGUCCGACGAUCAAAC; GUUCAGAGUUCUACAGUCCGACGAUCGGGC; GUUCAGAGUUCUACAGUCCGACGAUCUUUC; GUUCAGAGUUCUACAGUCCGACGAUCCCCC; GUUCAGAGUUCUACAGUCCGACGAUCACUC; GUUCAGAGUUCUACAGUCCGACGAUCGACC; GUUCAGAGUUCUACAGUCCGACGAUCUGAC; GUUCAGAGUUCUACAGUCCGACGAUCCUGC). The 5'-ligation reactions were separately in a 10% urea gel, and the region from 80-to-95 nucleotides for the RPFs and 80-to-105 nucleotides for the RNA input samples was excised, eluted, and precipitated from the gel. Gel-purified samples were reverse transcribed using SuperScript™ II Reverse Transcriptase (Invitrogen) with RTOligo (CAAGCAGAAGACGGCATACGA) according to the manufacturer (Invitrogen). Amplification was done using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) and oligos: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA and CAAGCAGAAGACGGCATACGA. PCR was carried out with an initial 30 s denaturation at 98°C, followed by 4 cycles of 30 s denaturation at 98°C, and 15 s extension at 72°C. Followed by 10- 15 cycles of 10 s denaturation at 98°C, 30 s annealing at 50°C, and 15 s extension at 72°C. Several reactions, with different numbers of cycles, were performed. Reactions were separated on a non-denaturing 8% polyacrylamide TBE gel, and DNA fragments of the correct size were extracted and sequenced using Illumina HiSeq2000.