title: GSM3034457: WT_S1P_2; Homo sapiens; RNA-Seq
library_construction_protocol: 1. Rinse cell monolayer with ice cold PBS once. Lyse cells directly in a culture dish by adding 1 ml of TRIZOL Reagent per 3.5 cm diameter dish and scraping with cell scraper. Pass the cell lysate several times through a pipette. Vortex thoroughly. The amount of TRIZOL reagent added is based on the area of the culture dish (1 ml per 10 cm2) and not on the number of cells present. An insufficient amount of TRIZOL Reagent may result in DNA contamination of the isolated RNA. 2. Add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent. Cap sample tubes securely. Vortex samples vigorously for 15 seconds and incubate them at room temperature for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 x g for 15 minutes at 4C. Following centrifugation, the mixture separates into lower red, phenolchloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. Transfer upper aqueous phase carefully without disturbing the interphase into fresh tube. 3. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIZOL Reagent used for the initial homogenization. Incubate samples at 15C for 10 minutes and centrifuge at not more than 12,000 x g for 10 minutes at 4C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube. 4. 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 no more than 7,500 x g for 5 minutes at 4C. Repeat above washing procedure once. Remove all leftover ethanol. 5. Air-dry or vacuum dry RNA pellet for 5-10 minutes. Do not dry the RNA pellet by centrifuge under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Dissolve RNA in DEPC-treated water by passing solution a few times through a pipette tip. https://support.illumina.com/content/dam/illumina-support/documents/myillumina/f5f619d3-2c4c-489b-80a3-e0414baa4e89/truseq_dna_sampleprep_guide_15026486_c.pdf
attributes.tag: GEO Accession
attributes.value: GSM3034457