GSM6043504: WAPL knockdown rep 1; Homo sapiens; OTHER
RNAi was performed as previously described. Following 72 h knockdown, cells were rinsed with Dulbecco's phosphate buffered saline (DPBS) and treated with trypsin to detach them from the plate. Cells were resuspended in cold supplemented McCoy's 5A media and three wells of a 6-well plate were pooled per replicate and placed on ice. From this point on, all steps were performed on ice, all buffers were pre-chilled, and samples were spun at 300xg for 10 min at 4ºC, unless otherwise noted. Cells were rinsed in PBS containing 1% Bovine Serum Albumin (BSA) to prevent cell clumping, and then resuspended in 1 ml Buffer W (10 mM Tris-HCl pH 8.0, 10 mM KCl, 250 mM sucrose, 5 mM MgCl2, 0.5 mM DTT, 10% glycerol, 1% BSA) then strained through a 35 μm nylon mesh filter. The tube was rinsed with an additional 1ml of Buffer W and passed through the same strainer. A 9X volume of Buffer P (Buffer W + 0.1% IGEPAL CA-630) was immediately added to each sample and nutated for 2 minutes at room temperature. Cells were resuspended in 500μl Buffer F (50 mM Tris-Cl pH 8.3, 40% glycerol, 5 mM MgCl2, 0.5 mM DTT, 1 μL/ml SUPERaseIn RNase inhibitor, 0.5% BSA) using a wide-bore P1000 tip and transferred to a low binding tube. The original tube was rinsed with another 500μl Buffer F and the samples were pooled. Samples were spun at 400xg and resuspended to 5 x 106 cells in 500μl Buffer F. Samples were flash frozen in liquid nitrogen and stored at −80°C PRO-seq library construction and data analysis was performed by the Nascent Transcriptomics Core at Harvard Medical School, Boston, MA. Aliquots of frozen (-80°C) permeabilized cells were thawed on ice and pipetted gently to fully resuspend. Aliquots were removed and permeabilized cells were counted using a Luna II, Logos Biosystems instrument. For each sample, 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/μl 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. 180μl 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 (NEBNextµltra 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.