GSM6189368: CT27_EVT_rep3; Homo sapiens; RNA-Seq
RNA was isolated with a TRIzol/chloroform precipitation followed by a cleanup with a RNeasy Mini Kit (74104, Qiagen, Germantown, MD). Frozen cell isolates in TRIzol were thawed on ice prior to starting the isolation. Once thawed, the samples were placed at room temperature for 5 min to promote dissociation of nucleoprotein complexes. Next, 200 μL of chloroform was added to each sample with vigorous shaking for 15 sec. Samples were placed at room temperature for 3 min before centrifugation at 12,000 g for 15 min at 4°C. After centrifugation, the upper, colorless, aqueous phase was removed and transferred to a new 1.5 mL tube and 1.5 volumes of 100% ethanol added. Isolates containing ethanol were mixed thoroughly by pipetting and 650 μl transferred to a RNeasy spin column. The protocol for the RNeasy Mini Kit was then followed to clean the sample post-TRIzol/chloroform extraction. RNA was then quantified using a Qubit RNA BR Assay Kit and RNA integrity assessed using RNA ScreenTape (5067-5577 and 5067-5576, Agilent) on the Agilent TapeStation platform (Agilent) prior to library preparation RNA libraries were prepared using a TruSeq Stranded RNA HT Sample Prep Kit (RS-122-2303, Illuminaon a Caliper Sciclone G3 platform (PerkinElmer, Waltham, MA) . Manual bead cleanup was performed using AMPure XP Beads (A63881, Beckman Coulter, Brea, CA) after llibrary preparation to remove primer-dimers. 1.0x volume of beads was added to the entire library volume. Samples were mixed by pipetting and incubated at room temperature for 5 min before being placed on a magnetic stand until the supernatant cleared. Once clear, the supernatant was removed and discarded, and the beads were washed twice with 80% ethanol while still on the magnet. Any residual ethanol was removed, and the beads were air-dried on the magnet for 5 min at room temperature. Once dry, 30 μL of Resuspension Buffer was added to each sample, the beads were resuspended by pipetting, and the solution incubated for 2 min at room temperature. Samples were then placed on a magnet until the supernatant was clear. The supernatant was then transferred to a new plate. A Qubit dsDNA BR Assay kit was used to determine the concentration of the library and a Fragment Analyzer Standard Sensitivity NGS Fragment Kit (DNF-473-10000, Agilent) was used to detect the size of the library and to verify removal of excess primer-dimers. Standard Illumina Free-Adapter Blocking was performed on the libraries. Cleaned, adapter-blocked libraries were loaded on a NovaSeq6000 (Illumina, San Diego, CA) with a run configuration of 151x8x8x151 and an average depth of 70M PE reads per library.