GSM7928542: Armstrong003_PR1044_Pcgf1_DMSO2; Mus musculus; OTHER
10 million cells were harvested and washed once with ice cold PBS (Gibco). To obtain single-cell suspensions, cell pellets were resuspended in 250mL wash buffer (10 mM Tris-Cl, pH 8.0, 10 mM KCl, 250 mM Sucrose, 5 mM MgCl2, 1 mM EGTA, 10 % (v/v) Glycerol, supplemented freshly with 0.5 mM DTT, 0.2mL/mL RNase inhibitor and protease inhibitor). 10mL of permealization buffer (10 mM Tris-Cl, pH 8.0, 10 mM KCl, 250 mM Sucrose, 5 mM MgCl2, 1 mM EGTA, 0.1 % (v/v), Igepal CA-630, 0.05 % (v/v) Tween-20, 10 % (v/v) Glycerol, supplemented freshly with 0.5mM DTT, 0.2mL/mL RNase inhibitor and protease inhibitor) was added dropwise and cells were incubated on ice for 5 min. Following centrifugation of the permeabilized cells, cells were washed in 10mL wash buffer and then resuspended in freezing buffer (50 mM Tris-Cl, pH 8.0, 40 % (v/v) glycerol, 5 mM MgCl2, 1.1 mM EDTA, supplemented freshly with 0.5 mM DTT and 1mL/mL RNase inhibitor). Cells were counted and permeabilization was visualized using trypan blue. At least 5e6 permeabilized cells were snap-frozen in 500ul freezing buffer in liquid nitrogen and stored at -80 C. 1 million permeabilized cells were used for nuclear run-on, with 50,000 permeabilized Drosophila S2 cells added to each sample for normalization. Nuclear run on assays and library preparation were performed essentially as described in Reimer et al. (Reimer et al., 2021)with modifications noted: 2X nuclear run-on buffer consisted of (10 mM Tris (pH 8), 10 mM MgCl2, 1 mM DTT, 300mM KCl, 40uM/ea biotin-11-NTPs (Perkin Elmer), 0.8U/uL SuperaseIN (Thermo), 1% sarkosyl). Run-on reactions were performed at 37°C. Adenylated 3' adapter was prepared using the 5' DNA adenylation kit (NEB) and ligated using T4 RNA ligase 2, truncated KQ (NEB, per manufacturers instructions with 15% PEG-8000 final) and incubated at 16°C overnight. 180uL of betaine blocking buffer (1.42g of betaine brought to 10mL with binding buffer supplemented to 0.6 uM blocking oligo (TCCGACGATCCCACGTTCCCGTGG/3InvdT/)) was mixed with ligations and incubated 5 min at 65°C and 2 min on ice prior to addition of streptavidin beads. After T4 polynucleotide kinase (NEB) treatment, beads were washed once each with high salt, low salt, and blocking oligo wash (0.25X T4 RNA ligase buffer (NEB), 0.3uM blocking oligo) solutions and resuspended in 5' adapter mix (10 pmol 5' adapter, 30 pmol blocking oligo, water). 5' adapter ligation was per Reimer but with 15% PEG-8000 final. Eluted cDNA was amplified 5-cycles (NEBNext Ultra II Q5 master mix (NEB) with Illumina TruSeq PCR primers RP-1 and RPI-X) following the manufacturer's suggested cycling protocol for library construction. A portion of preCR was serially diluted and for test amplification to determine optimal amplification of final libraries. Pooled libraries were sequenced using the Illumina NovaSeq platform.