GSM5268305: Satb1_cKO_rep1_RNAseq; Mus musculus; RNA-Seq
Freshly isolated thymocytes from female animals were resuspended in 1 ml of TRIzol Reagent (Invitrogen, 15596026) and RNA was isolated according to manufacturer's protocol. The aqueous phase with RNA was transferred into a tube and combined with 10 μg of Linear Acrylamide (Ambion, AM9520), 1/10 of sample volume of 3M CH3COONa (pH 5.2), 2.5 volumes of 100% Ethanol and tubes were mixed by flipping. Samples were incubated at –80°C for 40 minutes. Samples were centrifuged at 16,000 g, at 4°C for 30 minutes. The supernatant was removed and the pellet was washed twice with 75% Ethanol. The air-dried pellets were resuspended in 40 μl RNase-free water and incubated at 55°C for 15 minutes to dissolve RNA. To remove any residual DNA contamination, RNase-free DNase Buffer was added to the samples until 1x final concentration together with 20 units of DNase I (NEB, M0303L) and incubated at 37°C for 20 minutes. Samples were then purified using RNeasy Mini Kit (Qiagen, 74104) according to the manufacturer's protocol. RNA quality was evaluated using Agilent 2100 Bioanalyzer with Agilent RNA 6000 Nano Kit (Agilent Technologies, 5067-1511). Libraries were prepared using an Illumina TruSeq Stranded Total RNA kit with ribosomal depletion by Ribo-Zero Gold solution from Illumina according to the manufacturer's protocol and sequenced on an Illumina HiSeq 4000 (2x 75 bp).