Author: Ian M. Silverman; Sager J. Gosai; Nicholas Vrettos; Shawn W. Foley; Nathan D. Berkowitz; Zissimos Mourelatos; Brian D. Gregory
Title: Isolation and sequencing of AGO-bound RNAs reveals characteristics of mammalian stem-loop processing in vivo Document date: 2018_4_6
ID: 1pbshnw9_25
Snippet: Reactions were incubated at room temperature for 20 minutes. During this incubation, we seeded 6.0x10 5 HEK293T cells per replicate in two wells of a 6well plate (3.0x10 5 cells/well) in 2 mL media. We then added the siRNA mixture to each well dropwise, and allowed cells to incubate at 37ºC in 5% CO 2 for 48 hours. After 48 hours we repeated the transfection, harvesting the two wells per replicate (~1.2x10 5 cells) and dividing them into four we.....
Document: Reactions were incubated at room temperature for 20 minutes. During this incubation, we seeded 6.0x10 5 HEK293T cells per replicate in two wells of a 6well plate (3.0x10 5 cells/well) in 2 mL media. We then added the siRNA mixture to each well dropwise, and allowed cells to incubate at 37ºC in 5% CO 2 for 48 hours. After 48 hours we repeated the transfection, harvesting the two wells per replicate (~1.2x10 5 cells) and dividing them into four wells in a 6-well plate. We treated each well with 45 pmol of siRNAs, and allowed them to incubate at 37ºC in 5% CO 2 for another 48 hours. Cells were then pooled and washed with PBS prior to storage at -80ºC.
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