GSE211915
SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells [RNA-seq]
We performed gene expression profiling analysis using data obtained from RIP-seq inputs of 7 different cell lines. Overall design: ESCs were grow in wells of a 6-well plate to ~80% confluency. Cells were washed twice with 1x PBS and 1 mL of TRIzol was added per well. Samples were pipetted up and down at least 10 times, transferred to a microcentrifuge tube and briefly vortexed. Samples were incubated at RT for 5 minutes then 200µl of chloroform was added, samples were vortexed and incubated for 3 minutes at RT. Samples were spun down at 12000xg for 15 min at 4°C. The upper aqueous phase was moved to a new tube and 8µL of linear acrylamide (Thermo Fisher, AM9520) was added. Then 500µL of 100% isopropanol was added, samples were vortexed and incubated at RT for 10 minutes. Tubes were spun down 12000xg for 10 minutes at 4°C. Supernatant was removed and pellets washed with 1mL of cold 75% ethanol. Samples were briefly vortexed and spun down at 7500xg for 5 min at 4°C. Supernatant was discarded and pellet was dried by repeated spin down and aspiration. Final pellets were resuspended in 100µL water by pipetting up and down. For RIP-Seq inputs, 100ng of RNA prepared from RIP input samples was used for library preparations. For total RNA-Seq, 900ng of total RNA was used. Each library preparation included 1µL of 1:250 dilution of ERCC Spike-In RNAs (Ambion #4456653). 10µL total were prepped using the KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems; product #KR1351). Sequencing was performed on an Illumina Next-Seq 500, using high-output, 75-cycle kits.