title: GSM8854962: sci_L3_Strand_seq_yi284; Homo sapiens; OTHER
library_construction_protocol: Fixation and nucleosome depletion was performed as previously described in 'Methods and molecular design of sci-L3-WGS and sci-L3-target-seq', subsections 'Single Cell Preparation and Nucleosome Depletion' and 'Tagmentation (first-round barcodes) and ligation (second-round barcodes)' (Yin et al. 2019). Notably, cells were trypsinized and fixed with 37% formaldehyde (final 1.5% concentration) in 1x PBS at a cell density of 1 million/mL for 10 minutes at room temperature with gentle tube inversion. For first round barcoding,nuclei were distributed into individual wells. Any remaining nuclei were stained with DAPI at a final concentration of 5 ng/µL and used for FACS as previously described (Chovanec and Yin 2025). After the ligation reaction was stopped by the addition of the stop solution (lysis buffer (LB: 60 mM Tris-Ac pH 8.3, 2 mM EDTA pH 8.0, 15 mM DTT) with 0.1% Triton-X100 (LBT)), nuclei were pooled and stained with Hoechst-33258 to a final concentration of 5 ng/µL and the quenched population was sorted (100-300 nuclei per well) into a 96-well plate containing 3 µL of LB. Right after gap extension with the Bst WarmStart 2.0 polymerase at 68°C for 5 minutes and its inactivation with 80°C for 10 minutes, Hoechst-33258 was added at a final concentration of 10ng/µL and incubated at room temperature for 10 minutes in the dark. A 1 or 5 minute exposure in a Bio-Rad Gel Doc with a 365nm UV bulb, or a dose of 27-4000 mJ/cm2 was administered with a UVP crosslinker (CL-3000L) as previously described (Chovanec and Yin 2025). At this stage, each well had a volume of around 7.7 µL, to which 0.3uL of USER enzyme (a mix of uracil DNA glycosylase and endonuclease VIII, referred to as UDG+EndoVIII in the text, NEB) was added and incubated at 37°C for 15 minutes. Next, 0.9 µL of its inhibitor UGI (NEB) was added with a further incubation at 37°C for 10 minutes. Finally, the T7 in vitro transcription system was assembled by adding 2 µL H2O, 2 µL T7 Pol mix and 10 µL rNMP mix (NEB, HiScribe T7 Quick High Yield RNA Synthesis Kit) and incubated at 37°C for 10-16 hours. The remaining section, 'RNA purification, RT and SSS', was performed exactly as previously described (Yin et al. 2019).