GSM4676525: BglHalo_Xist_SLAM-seq_60min; Mus musculus; RNA-Seq
After cells were collected by centrifugation at 1 500 g for 5 min in PBS, nuclear extracts were obtained as follows: Cells were washed again with PBS and resuspended in 10 volumes buffer A (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and complete protease inhibitors in RNase free water). Subsequently cells were incubated on ice for 10 min and recovered by centrifugation, followed by resuspension in 3 volumes buffer A with 0.1 % NP-40 for 10 min. Nuclei were recovered by centrifugation at and TRIzol/chloroform RNA extraction was performed using the SLAMSeq Kinetics Kit (Lexogen). Then, samples were treated with Iodoacetamide to modify the 4-thiol group of S4U-containing nucleotides via the addition of a carboxyamidomethyl group using the SLAMSeq Kinetics Kit (Lexogen). The RNA was precipitated and washed again, before being resuspended in nuclease free water. 1 ng RNA of each sample were run on the bioanalyzer to determine the exact RNA concentration and RNA integrity of all samples. Based on this, 1 μg RNA of each sample was then taken forward for library preparation using the Illumina TruSeq stranded total RNA kit (RS-122-2301). Quantification of the libraries was performed by qPCR using KAPA Library Quantification DNA standards (Kapa Biosystems, KK4903). Finally, the libraries were pooled and 2 × 81 paired-end sequencing was performed using Illumina NextSeq500 (FC-404-2002). TruSeq RNAseq