title: GSM7750395: mESCs_scGROseq_Exp160_c04; Mus musculus; OTHER
library_construction_protocol: For nuclei isolation, the pellet was resuspended in ice-cold douncing buffer (10 mM Tris-Cl pH 7.4, 300 mM Sucrose, 3 mM CaCl2, 2 mM MgCl2, 0.1 % Triton X-100, 0.5 mM DTT, 0.1x Halt Protease inhibitor, and 0.02 U/ul RNase inhibitor) and transferred to a 7 ml dounce homogenizer. After incubation on ice for 5 minutes, the cells were dounced 25 times with a tight pestle, transferred back to the 15 ml conical tube, and centrifuged to pellet the nuclei. The pellet was washed twice in douncing buffer. For cell permeabilization, the pellet was resuspended in ice-cold permeabilization buffer (10 mM Tris-Cl pH 7.4, 300 mM Sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.05 % Tween-20, 0.1% NP-40, 0.5 mM DTT, 0.1x Halt Protease inhibitor, and 0.02 U/ul RNase inhibitor). After incubation on ice for 5 minutes, the cells were centrifuged to pellet the nuclei. The pellet was washed twice in the permeabilization buffer. A 96-well plate containing 5'-AzScBc DNA with a unique cell barcode in each well previously synthesized and aliquoted is thawed from -80°C. Sodium Ascorbate, PEG 8000, CuSO4, and accelerating ligand BTTAA was prepared and dispensed into each well of the 96-well plate containing 5'-AzScBc DNA. The CuAAC reaction mix was dispensed into individual wells containing single cells in Urea using a multi-channel pipette or a 96-well pipettor. The 96-well plates are sealed, vortexed for 10 seconds in an orbital vortexer, and centrifuged for 1 min at 500 g prior to incubation for 2 hours at 50°C. After completion of incubation, the CuAAC reaction was quenched with 5 mM EDTA and pooled from 96-wells into a single 1.5 ml eppendorf tube. PEG 8000 was removed with Trizol. Remaining CuAAC reagents (Sodium Ascorbate, CuSO4, and mM BTTAA) were removed with a centrifugal filter with 3 kDa cellulose membrane (Amicon, 2020-04). The purified RNA was fragmented with 10 mM ZnCl2 for 5 min at 65°C. Reverse transcription of the clicked RNA-DNA conjugate was performed Reverse Transcriptase lacking RNase H activity but capable of RNA-dependent and DNA-dependent polymerase activity, non-templated addition and template switching. RT reaction was incubated with RNA-DNA conjugate for 2 hours at 50°C. The cDNA was size-selected in 10% denaturing polyacrylamide gel electrophoresis (PAGE) away from the unclicked 5'-AzScBc DNA and empty cDNA formed between the 5'-AzScBc DNA and TSO. library was PCR amplified with Q5 High-Fidelity 2X Master Mix (NEB, M0492S) and 0.5 uM PCR primers with unique dual index using the two-step PCR cycles.