GSM5057852: CB cells - control-FC_2; Mus musculus; RNA-Seq
After cell culture incubation medium was removed and cells were washed with cold 1x PBS (pH 7.4). Total of 500 µL of cold nuclei buffer (10 mM Tris-HCl pH 7.9; 100 mM KCl 5 mM MgCl2) was added after removing PBS and incubated for 5 min on ice. About 20 µL of 10% NP40 and 0.5 µL DTT were added and were shaken at 200 rpm for 2 min on ice to perform cell lysis. Suspension was transferred to a 1.5-mL tube and centrifuged for 2 min at 1200_x0002_ g at 4 °C. After discarding of supernatant, 500 µL of nuclei buffer was added for pellet resuspension. After centrifugation for 2 min at 200 g at 4 °C, supernatant was discarded. Pellet was washed twice with 125 µL of glycerol buffer (20 mM Tris-HCl pH 7.4; 50% glycerol; 75 mM KCl; 1 mM DTT; 5 mM MgCl2) with centrifugation for 5 min at 500_x0002_ g at  4 °C. Finally, supernatant was discarded and pellet resuspended in 50 µL of nuclei buffer. After adding 450 µL of Trizol®Reagent (Thermo Fisher Scientific, Waltham, MA, USA) all samples were snap frozen on dry ice and stored at 80 °C. For RNA-sequencing the samples were subjected to standard RNA isolation procedure using Trizol®Reagent with additional application of GlycoBlue (Thermo Fisher Scientific, Waltham, MA, USA) to a final concentration of 0.2% during RNA precipitation for better visualization of the pellet. RNA and DNA quantities were determined using Qubit 4 Fluorometer (Thermo Fisher Scientific,Waltham, MA, USA). Library preparation for RNA-Seq was performed using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA, E7760) starting from 36 ng of total RNA as input. Ribosomal depletion was achieved by using the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (New England Biolabs, Ipswich, MA, USA, E6310) and indexing was performed by the use of NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, Ipswich, MA, USA, E7335). Accurate quantification of the input RNA was performed with the QuantiFluor® RNA System (Promega, Madison, WI, USA, E3310) and by TapeStation RNA ScreenTape Analysis (Agilent, Santa Clara, CA, USA, 5067-5576). Accurate quantification of cDNA libraries was performed by using the QuantiFluor™ dsDNA System (Promega, Madison, WI, USA). The size range of final cDNA libraries was determined applying the TapeStation D1000 ScreenTape (Agilent, Santa Clara, CA, USA, 5067-5582). cDNA libraries were amplified and sequenced using the NextSeq 500 with NextSeq 500/550 High Output Kit v2.5 (150 cycles; Illumina, San Diego, CA, USA, 20024907). Sequencing was performed as paired end; 2 _x0002_ 76 bp; single indexing with ~30 million reads per sample. total RNA-Seq (nuclear enriched RNA isolation, ribosomal depletion)