GSM8615499: sci_L3_Strand_seq_yi293; Homo sapiens; OTHER
sci-L3-Strand-seq. 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, Yi et.al. 2019 PMID: 31495564). Notably, cells were trypsinized and fixed with 37% formaldehyde (final 1-1.5% 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, Patski nuclei were distributed into wells with barcodes 1-12, while HAP1 nuclei were distributed into wells with barcodes 13-24. The remaining nuclei were stained with DAPI at a final concentration of 5 mg/mL and used for FACS as described in Fig2A. 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 10ng/µL and the quenched population was sorted (100-300 nuclei per well) into a 96-well plate containing 3 µL of LB. The major differences of sci-L3-Strand-seq arise in the 'Cell lysis, gap extension and linear amplification by in vitro transcription' subsection of the protocol (1). 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). Note, we did not observe any differences in library quality between 1 or 5 minute exposures, or in doses above 270 mJ/cm2. 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 as previously described 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, Yi et.al. 2019 PMID: 31495564).  sci-L3-Hi-C. The major differences of sci-L3-Hi-C arise in the first 'Single Cell Preparation and Nucleosome Depletion' subsection of the protocol (1). After 2% formaldehyde fixation and quenching as done for sci-L3-Strand-seq, the proximity ligation was performed largely as previously described for Dip-C (18). The pelleted cells after fixation were washed with ice-cold 1x PBS, resuspended in 500 µL Hi-C lysis buffer (10mM Tris pH8.0, 10mM NaCl, 0.2% IGEPAL with 100uL Protease inhibitors (“PI”, Sigma P8340) and incubated on ice for at least 15 minutes. The nuclei were pelleted at 2500g for 5 minutes at 4°C and washed with ice-cold Hi-C lysis buffer. The pellet was resuspended in 50 µL of 0.5% SDS (diluted with H2O) and incubated for 10 minutes at 62°C. The SDS was quenched with the addition of Triton X-100 (145 µL H2O, 25 µL 10% Triton X-100) and with a further incubation of 15 minutes at 37°C. Next, 25 µL of NEBuffer2 was added, followed by 20 µL of 25U/µL MboI (NEB, R0147M) and left overnight at 37°C. The nuclei were pelleted at 2500g for 5 minutes at 4°C and washed with 1 mL of ligation buffer (1x T4 DNA ligase buffer, NEB B0202S with 0.1mg/mL BSA, NEB B9000S). Ligation was performed with 1 mL ligation buffer and 10 µL of 1U/µL T4 DNA ligase (Life Tech 15224-025) at 16°C for 4 hours. Nuclei were passed through a 35 µM cell strainer, pelleted at 2500g for 5 minutes at 4°C, washed with 1 mL lysis buffer (LB: 60 mM Tris-Ac pH 8.3, 2 mM EDTA pH 8.0, 15 mM DTT) and resuspended with LB at a concentration of 20,000 nuclei per µL. The remaining steps were performed exactly as previously described in subsection 'Tagmentation (first-round barcodes) and ligation (second-round barcodes)'  onwards (Yin, Yi et.al. 2019 PMID: 31495564).  sci-L3-RNA/ATAC. The major differences of sci-L3-RNA/ATAC arise in the nucleosome depletion step previously described in the 'Methods and molecular design of sci-L3-RNA/DNA co-assay' subsection of the protocol (Yin, Yi et.al. 2019 PMID: 31495564). Cells were trypsinized, combined together (HEK293T, BJ-5ta, NIH/3T3), and fixed with 2% PFA in 1x PBS at room temperature for 10 minutes at a density of 1 million/mL. The subsequent quenching (with glycine), washing, and nuclei isolation (with 0.1% IGEPAL) steps are identical with sci-L3-WGS, except the addition of 1% Superase-In to all the LB and 1xNEBuffer2.1 buffers. After the isolation of nuclei, the pellet was split in half. One half subjected to nucleosome depletion with the addition of 776 µL of 1x NEBuffer2.1, 24 µL of 10% SDS and an incubation at 42°C for 15 minutes. The SDS was quenched with 180 µL of 10% Triton-X100 with 10 µL Superase-In, and further incubated at 42°C for 10 minute. The depleted nuclei were pelleted, washed with 1 mL LB with 1% Superase-In, and resuspended in LB with 1% Superase-In at a 20,000 nuclei per µL concentration. These nuclei represent the sci-L3-RNA/DNA sample. For the other half, the nuclei were washed with 200 µL of LB with 1% Superase-In and 0.1% Triton-X100 and resuspended in LB with 1% Superase-In at a 20,000 nuclei per µL concentration. These nuclei represent the sci-L3-RNA/ATAC sample. For the first round barcoding, the nucleosome depleted nuclei were distributed into wells with barcodes 1-5, while the non-nucleosome depleted nuclei were distributed into wells with barcodes 6-10. The subsequent steps were performed exactly as previously described in the sci-L3-RNA/DNA co-assay subsections 'RT and Tagmentation, Ligation, FACS, and Cell Lysis', 'Gap Extension and Linear Amplification by In Vitro Transcription', and 'RNA Purification, RT, and SSS' (Yin, Yi et.al. 2019 PMID: 31495564).