GSM3581951: r26_x5_3D_nuc_R1; Mus musculus; RNA-Seq
All steps were performed at 4°C. Cells were washed twice with 1mL cold PBS, scraped in 1mL PBS, centrifuged at 1500xrcf for 5min, and resuspended in 250µL low salt solution (10mM KCl, 1.5mM MgCl2, 20mM Tris-HCl pH 7.5) supplemented with 1mM PMSF, 1mM DTT, and 1:100 protease inhibitor cocktail (PIC; Sigma P8340). Triton X-100 was added to a final concentration of 0.1% and cells were rotated for 10min, then centrifuged for 5min at 1500xrcf. 200µL of supernatant was added to 1mL Trizol (Invitrogen; cytosolic fraction). The remaining supernatant was discarded and the nuclear pellet was washed by rotating for 2min in low salt solution without Triton X-100 and centrifuged at 1300rpm for 10min. Nuclear pellets, which at this stage contained both soluble nuclear and chromatin-bound fractions, were resuspended in 1mL Trizol. Isolation of RNA from Trizol was performed according to manufacturer protocol. RNA-seq libraries were prepared using the RNA HyperPrep Kit with RiboErase (Kapa Biosciences) and sequenced on an Illumina NextSeq 500 machine using a 75-cycle high output NextSeq kit (Illumina).