GSM8690469: PRO-seq_Ctrl2; Homo sapiens; OTHER
Cells were rinsed with room temperature 1X PBS, detached using trypsin (quenched with ice-cold DMEM + 10% FBS), and collected in 50 mL conical tubes. The cells were pelleted by centrifugation, washed with  cold 1X PBS, and centrifuged again under the same conditions. The cell pellets were then resuspended in wash buffer (10 mM Tris-Cl, 10 mM KCl, 250 mM Sucrose, 5 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 10 % Glycerol) to achieve a single-cell suspension, followed by the addition of 10 mL permeabilization buffer  (wash buffer with 0.1 % (v/v) Igepal CA-630 and 0.05 % Tween-20). After 5 minutes of incubation on ice for permeabilization, cells were centrifuged at 400 × g for 8 minutes at 4°C, and the supernatant was removed. The pellets were washed twice with wash buffer and finally resuspended in 200 µL of freeze buffer (50 mM Tris-Cl, 40 % (v/v) glycerol, 5 mM MgCl2, 1.1 mM EDTA, 0.5 mM DTT), followed by an additional rinse with 200 µL freeze buffer, to yield a total of 400 µL. The cells were snap-frozen in liquid nitrogen and stored at -80°C until further processing. Libraries were prepared and sequenced by Nascent Transcriptomics Core at Harvard Medical School as described in Mimoso et al. 2023; https://doi.org/10.1002/cpz1.961