GSM7749098: HUES66 WT RNA; Homo sapiens; RNA-Seq
Total RNA was extracted using TRIzol (Invitrogen #15596018) according to the manufacturer's protocol.  To isolate RNA from cytosolic and nuclear fractions, cells were washed twice with 1mL cold PBS and scraped in 1mL PBS + 1mM PMSF + 1:100 protease inhibitor cocktail (PIC, Sigma P8340). 200uL was removed at this step and added to 1mL Trizol (total RNA). The remaining cells were centrifuged at 1500xrcf. for 5 min, and resuspended in 250uL low salt solution (10mM KCl, 1.5mM MgCl2, 20mM Tris-HCl pH 7.5) supplemented with 1mM PMSF, 1mM DTT, and 1x PIC. Triton X-100 was added to a final concentration of 0.1% and cells were rotated for 10 min at 4C, then centrifuged for 5 min at 1500xrcf. 200uL of supernatant was removed and added to 1mL Trizol (cytosolic fraction). The remaining supernatant was discarded and the nuclear pellet was washed by rotating for 2min at 4C in low salt solution without Triton X-100 and centrifuged at 1300rpm for 10 min. Nuclear pellet was resuspended in 1mL Trizol (nuclear fraction). Isolation of RNA from Trizol was performed according to the manufacturer protocol. Per RNA sample, we combined 1 µg of total RNA + 2 µl 1:100 ERCC RNA Spike-in Mix #1 (Invitrogen #4456740). Libraries were prepared using the Kapa RNA HyperPrep Kit with RiboErase (Roche #08098131702), pooled, and sequenced using the Nextseq 500/550 High Output Kit with 150 cycles (Illumina #20024907).