GSM5833937: E7_dTAG47_12h; Homo sapiens; OTHER
PRO-seq was performed as described (Liu et al. 2021). Five million HUDEP-2 were harvested on ice, and washed with cold PBS. Cells were resuspended in 2 mL cold Buffer W (10 mM Tris-Cl, pH 8.0, 10 mM KCl, 250 mM sucrose, 5 mM MgCl2, 0.5 mM DTT, 10% glycerol, 0.5% BSA) using wide-bore P1000 tip and transferred to a 2 mL low binding tube and strained through a 35 micron nylon mesh cell strainer. Immediately add 18 mL Chilled Buffer P (Buffer W supplemented with 0.05% Igepal CA630) to cell suspension and mix gently for 2 min. Cells were centrifuged at 700 x g for 8 min and resuspended in 1 mL Buffer F (50 mM Tris-HCl pH 8.3, 40% glycerol, 5 mM MgCl2, 0.5 mM DTT, 0.5% BSA), and centrifuged again at 700 x g for 8 min. The pellets were resuspended in 400 μL Buffer F and counted using a hemocytometer to ensure that >95% cells were permeabilized. The cell density was adjusted to 1 x 106/100 μL, and the suspension was snap-frozen and stored at -80°C. cDNA was immediately amplified for 5-cycles ("preCR" step).  A PCR cocktail consisting of NEBNext Ultra II Q5 master mix (NEB) and Illumina TruSeq PCR primers (RP-1, common; and RPI-X, indexing) was added to each cDNA and amplification carried out according to manufacturer's suggested 2-step cycling conditions for NGS applications. To determine optimal library amplification, the preCRs were thawed on ice and an aliquot serially diluted. Each dilution was mixed with a PCR cocktail (Q5 DNA polymerase with optional high GC enhancer) containing 10 mM/ea of Illumina universal P5 and P7 primers and amplified 15-cycles.  The reactions were electrophoresed on a 2.2% agarose gel (SeaKem) cast with SYBR gold (Thermo) and analyzed to determine the optimal number of cycles for final amplification. preCRs were transferred from ice to a pre-heated thermal cycler block and amplified for the appropriate number of additional cycles to reach the total determined in the test amplification. Pooled libraries were sequenced using an Illumina NovaSeq S1 (PE 50), Novaseq S4 (200 cycle kit), NextSeq in high output mode (PE 75), NovaSeq S1 with 100 cycle kit (PE50).