GSM4708424: SLAMseq_ESC_wt_dTag_2h_SLAM_r3; Mus musculus; RNA-Seq
Cells were lysed directly in TRIzol (500 μl, Invitrogen). Samples were processed according to the SLAMseq Kinetics Kit – Anabolic Kinetics Module (Lexogen). Briefly, added 100 µl chloroform:isoamyl alcohol mix (24:1) to TRIzol lysate, shake tube vigorously (15 sec), incubate at RT for 3 min. Centrifuge at (16,000g, 15 min, 4°C) Transfer the aqueous phase to a new tube. Measure the volume of the aqueous phase using a pipette. Add 1 µl of Carrier Substance, 1/1,000th volumes of Reducing Agent, and 1 volume of 2-propanol. Vortex well. Incubate for at RT (10 min) and centrifuge at (16,000g, 20 min, 4°C). Wash pellet with 500 µl 75 % EtOH and 0.5 µl of Reducing Agent. Vortex well and centrifuge (7,500g, 5 min, 4°C). Remove supernatant, let pellet dry for 5-10 min, and resuspend it in 16 µl of Elution Buffer. Incubate for 10 min, 55°C and measure the concentration by NanoDrop. Prepare a mastermix containing 5 µl of freshly prepared 100 mM Iodoacteamide, 25 µl of Organic Solvent, and 5 µl of Sodium Phosphate per sample. Sequencing libraries were prepared from 100ng of purified total RNA for three biological replicates using TruSeq stranded Total RNA Library Prep (Illumina).
GEO Accession
GSM4708424