| Extract Protocol: |
For RNA-seq, cells were harvested with Tri-Reagent (Sigma-Aldrich), total RNA was extracted. poly-A selection was performed using Dynabeads mRNA DIRECT Purification Kit (Invitrogen). For ribosome profiling, medium was aspirated from dishes, which were immediately placed on ice and rinsed with 10 ml ice-cold PBS supplemented with drugs used in pretreatment of the cells. PBS was aspirated and 800 μl ice-cold lysis buffer (20 mM Tris, pH 7.4, 250 mM NaCl, 15 mM MgCl2, 1 mM dithiothreito, 0.5% Triton X-100 and 24 U / ml Turbo DNase (Ambion, AM2239), along with any drugs used for sample treatment) was dripped onto dishes. Cells were scraped and the lysate was removed and incubated 10 min on ice. The lysate was then clarified by centrifugation for 10 min at 20,000 × g, 4°C and ∼1.1 ml supernatant was recovered. For SLAM-seq libraries Cells were harvested with Tri-reagent at 1, 2, 3, and 4 hours after addition of 4sU (corresponding to 4,5,6 and 7hpi for infected cells) . RNA was extracted under reducing conditions and treated with Iodoacetamide. poly-A selection was performed using Dynabeads mRNA DIRECT Purification Kit (Invitrogen). For fractionation, first a fraction of 10% of the cells was transferred to a new tube and RNA was extracted in Tri-reagent to obtain whole cellular extract. Nuclear fraction was separated from cytoplasmic fraction by centrifugation following cell lysis. RNA was then extracted from both fraction in Tri-reagent. poly-A selection was performed using Dynabeads mRNA DIRECT Purification Kit (Invitrogen).
RNA-seq: mRNA samples were subjected to DNAseI treatment and 3’ dephpsphorylation using FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) and T4 PNK (NEB) followed by 3’ adaptor ligation using T4 ligase (NEB). The ligated products were then used for reverse transcription with SSIII (Invitrogen) for first strand cDNA synthesis. The cDNA products were 3’ ligated with a second adaptor using T4 ligase and amplified for 8 cycles in a PCR for final library products of 200-300bp. Ribo-seq: Cells were treated with 100µg/mL CHX for 1 minute. Cells were then placed on ice, washed twice with PBS containing 100µg/mL CHX, scraped from 10cm plates, pelleted and lysed with lysis buffer (1% triton in 20mM Tris 7.5, 150mM NaCl, 5mM MgCl2, 1mM dithiothreitol supplemented with 10 U/ml Turbo DNase and 100µg/ml cycloheximide). After lysis samples stood on ice for 2h and subsequent Ribo-seq library generation was performed. Briefly, cell lysate was treated with RNAseI for 45min at room temperature followed by SUPERase-In quenching. Sample was loaded on sucrose solution (34% sucrose, 20mM Tris 7.5, 150mM NaCl, 5mM MgCl2, 1mM dithiothreitol and 100µg/ml cycloheximide) and spun for 1h at 100K RPM using TLA-110 rotor (Beckman) at 4c. Pellet was harvested using TRI reagent and the RNA was collected using chloroform phase separation. For size selection, 15uG of total RNA was loaded into 15% TBE-UREA gel for 65min, and 28-34 nt footprints were excised using 28 nt and 34 nt flanking RNA oligos, followed by RNA extraction and ribo-seq protocol (Finkel Y. et. al., 2020, ELife)
RNA-seq of whole cells as well as nuclear and cytoplasmic fractions, ribosome profiling, and SLAM-seq. |
| Library Construction Protocol: |
cell lysate was treated with RNAseI for 45min at room temperature followed by SUPERase-In quenching. Sample was loaded on sucrose solution (34% sucrose, 20mM Tris 7.5, 150mM NaCl, 5mM MgCl2, 1mM dithiothreitol and 100µg/ml cycloheximide) and spun for 1h at 100K RPM using TLA-110 rotor (Beckman) at 4c. Pellet was harvested using TRI reagent and the RNA was collected using chloroform phase separation. For size selection, 15uG of total RNA was loaded into 15% TBE-UREA gel for 65min, and 28-34 nt footprints were excised using 28 nt and 34 nt flanking RNA oligos, followed by RNA extraction and ribo-seq protocol (Finkel Y. et. al., 2020, ELife) |
