title: GSM2879589: Control Cells; Homo sapiens; OTHER
library_construction_protocol: HEK293T cells and HEK293T stable cells expressing EBNA1-FLAG-HA were grown upon confluency in P150 dishes, harvested and pelleted. They were then washed twice in cold PBS, and resuspended in the same volume as the pellet of polysome lysis buffer (10 mM Hepes pH 7.0, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT, 100 u/mL RNase inhibitor and complete protease inhibitor (Roche)). After an incubation of 5 minutes on ice, they were frozen at -80°C to complete the lysis. After a rapid thaw, tubes were centrifuged 20 minutes at 4°C, 13 000g. The supernatant was dosed using the Bradford assay (Thermo Scientific Coomassie Protein Assay) in triplicate. For each IP, 50 uL of Anti-Ha matrix (Roche) was centrifuged 1 min at 13,400 rpm and the supernatant was discarded. Beads were washed with 1 mL of NT2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40) followed by 1 min centrifugation at 13,400 rpm for 5 times. They were then resuspended in 900 uL of NET-2 buffer (NT2 buffer supplemented with 20 mM EDTA, pH 8.0, 1 mM DTT and 100 u/mL RNase inhibitor) and 100 uL (adjusted with NT-2 buffer to a total protein quantity of 2.5 mg) of lysate from either control or EBNA1 expressing cells was added. Tubes were inverted a couple of times, centrifuged 1 min at 13,400 rpm, 4°C and 100 uL was removed as the input to evaluate RNA degradation. All tubes were incubated on a rotating wheel at 4°C overnight. Beads were precipitated at 5000g for 5 min, 4°C and washed five times with ice-cold NT2 buffer. Upon the last washing, beads were resuspended in 90 uL of NT-2 buffer and 10 uL of RQ1 DNase and incubated at 37°C for 30 minutes. Input tubes were directly added 10 uL of RQ1 DNase. Dnase-treated IP were diluted with 1 mL NT-2 buffer, centrifuged and supernatant was discarded. Beads were resuspended in 150 uL of proteinase K buffer (1% SDS, 1.2 mg/mL proteinase K from Boehringer Mannheim), and input tubes were directly added SDS and proteinase K to the same volume. Tubes were incubated at 55°C during 30 minutes with inversion at every 10 minutes. RNA was then extracted with phenol-chloroform, followed by a second chloroform extraction step. Precipitation of RNA using 272 mM ammonium acetate, 122 mM LiCl and 27 ug/mL glycogen in ice-cold ethanol was carried out during 2 hours at -80°C and followed by high-speed centrifugation. Pellets were washed in 75% ethanol, ethanol was thoroughly removed and RNA was resuspended in water. Quality (input) and quantity (input and IP) assessments were performed on Agilent Nano Chip (Catalog number 5067-1511). Inputs from both control and EBNA1 expressing cells both showed good RNA integrity (RIN = 8.9 and 9.2, respectively), thus underlining RNA was not deteriorated due to experimental procedures. RNA was ribodepleted using Illumina Ribo-Zero rRNA Removal Kit as per manufacturer's protocol. The RNA-seq library was then built using Illumina SSV21106 kit from 9 l ribo-depleted RNA. Library quality was assessed using Agilent DNA HS Chip (Catatalog number 5067-4626). Library quantification was performed by qPCR following Illumina Kappa library quantification protocol. HEK293T and HEK293T-EBNA1 libraries were multiplexed and sequencing was done using Illumina HiSeq 4000 at 100bp paired-end reads at McGill University and Génome Québec Innovation Centre Sequencing Service.
attributes.tag: GEO Accession
attributes.value: GSM2879589