GSM2042347: RNAseq Knockout Satb2 Replicate5; Mus musculus; RNA-Seq
Each sample was supplied with 500µl Trizol-reagent and 10 ceramic beads before homogenizing in an Omni Bead-Ruptor 24 (Omni International; Kennesaw, USA) for 10s at a relative speed of 2.6. Afterwards, the samples were incubated at RT for 5 minutes to ensure a proper lysis of the tissue. 100µl chloroform was added, samples were mixed manually for 15 seconds andincubated for another 5 minutes at RT. After centrifuging at 12,000g for 15 minutes at 4o C, the transparent upper phase was transferred to a fresh tube. 250µl isopropanol and 1µl GlycoBlue (Thermo Fisher Scientific; Waltham, USA) were added to each sample before they were incubated at -20o C over night to precipi- tatethe RNA. Then, the samples were centrifuged at 12,000g for 30 minutes at 4o C and the supernatant was discarded carefully. For washing, the RNA pellets were mixed with 300µl 75% ethanol, vortexed and centrifuged at 12,000g for 5 minutes at 4oC before removing the supernatant. This washing step was repeated once. Before redissolving the RNA, the tubes were briefly spun and freed from remaining liquid. Samples were air-dried for 2 minutes to clean the RNA from ethanol. Finally, RNA pellets were redissolved in sequencing graded water and incubated for 10 minutes at 42oC before proceeding with DNAse treatment Standard RNAseq and smallRNAseq library preperation
GEO Accession
GSM2042347