title: N1Esf6L2
organism: Homo sapiens
description: Protocols: Total RNA extraction Total RNA was isolated from MCF7 monoculture, 6 and 19-day old MCTS by a TRIzol (Invitrogen, CA, USA) adapted protocol. Particular adjustments previous to the TRIzol extraction were made for both culture conditions. In the monoculture, we used a 70-80% confluent culture, then we poured 1 mL of TRIzol directly to the culture dish and homogenized until all cells were detached from the dish. This step was assisted by the use of a scraper. In the 6-day and 19-day old MCTS culture, the MCTS were retrieved by centrifugation and resuspended in 1 mL of TRIzol. The solution was homogenized vigorously until there were no cellular lumps. The subsequent steps described below were used for both culture conditions. We added 10 µL of triton 2% (Merck T8787, St. Louis, USA) and incubated for 10 min. at room temperature. Next, we added 200 µL of chloroform, mixed through inversion and incubated for 5 min. at room temperature. Tubes were centrifuged at 12,000 rpm during 15 min. at 18 ºC. The aqueous phase was retrieved, we added 0.25 µL of glycogen as co-precipitation reagent (ThermoFisher R0561, Waltham, USA) and isopropanol was added in a 1:1 ratio (regarding TRIzol). The mixture was homogenized gently, incubated for 20 min. at −20 ºC and centrifuged at 13,000 rpm during 45 min. at 18 ºC. Finally, the pellet was washed three times with ethanol 75%, dissolved in ultrapure DDW (not treated with DEPC) and stored at −80 °C. The library construction was made by following the manufacturer procedure of TruSeq Stranded Total RNA with Ribo Zero Human/Mouse/Rat.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-11-11T04:07:40Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-11-06T11:54:00Z
attributes.tag: External Id
attributes.value: SAMEA7536472
attributes.tag: INSDC center name
attributes.value: INMEGEN
attributes.tag: INSDC first public
attributes.value: 2020-11-11T04:07:40Z
attributes.tag: INSDC last update
attributes.value: 2020-11-06T11:54:00Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9741:N1Esf6L2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: MCF-7
attributes.tag: cell type
attributes.value: breast invasive ductal carcinoma cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: breast cancer
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: multicellular tumor spheroid culture
attributes.tag: organism part
attributes.value: breast
attributes.tag: sample name
attributes.value: E-MTAB-9741:N1Esf6L2
attributes.tag: scientific_name
attributes.value: Homo sapiens