title: GSM2837387: iNSC_3D_Ctrl_R2; Homo sapiens; RNA-Seq
library_construction_protocol: For 2D cells; Human fetal cortical cells were grown in monolayer. Firstly we rinsed cell monolayer with ice cold PBS once. WE add 500 ul of TRIZOL reagent to lyse cells directly in a 24 well culture dish and scrape with a cell scraper. Pass the cell lysate several times through a pipette and then we vortex thoroughly. Add 0.2 ml of chloroform per 1 ml of TRIZOL. Vortex samples for 15 seconds and incubate them at room temperature for 2 to 3 minutes. Centrifuge the samples at 12,000 x g for 15 minutes at room temperature. Transfer upper aqueous phase into fresh RNAse-free tube. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIZOL used for the initial homogenization. Incubate samples at room temperature for 10 minutes and centrifuge at 12,000 x g for 10 minutes at 4oC. Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of TRIZOL Reagent used for the initial homogenization. Mix the samples by vortexing and centrifuge at 7,500 x g for 5 minutes at 4oC. Repeat above washing procedure once. Remove all leftover ethanol. Air-dry or vacuum dry RNA pellet for 5-10 minutes. Dissolve RNA in DEPC-treated water by passing solution a few times through a pipette tip. For cells from 3D gels, we used Norgen Total RNA Isolation Kit. RNA isolation performed according to the protocol suggest by the company except; 3 gels lysed in 1ml RL buffer with 10 µL beta-mercaptoethanol. Centrifuged at 10000g for 5 min at RT and supernatant (~930 µL) transfered to a new eppendorf. 580 µL %96-99 ethanol added and mixed throughly. After passing through samples on columns, washings and DNase I treatment done accroding to the protocol suggests and RNA eluted in50 µL  nuclease free water. Samples ran on Agilent Bioanalyser for RNA quality, samples with RNA integrity >= 9.0 used for library preparation. Total RNA with an integrity number of ≥ 8 was used. mRNA was isolated from total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module. After chemical fragmentation by incubating for 15 min at 94°C the sample was directly subjected to the workflow for strand specific RNA-Seq library preparation (Ultra Directional RNA Library Prep, NEB)
attributes.tag: GEO Accession
attributes.value: GSM2837387