GSM5151360: PD196-1_cells-vs-nuclei-vs-fixednuclei; Homo sapiens; Mus musculus; OTHER
Fresh nuclei isolation: A total of 5 million cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifugation (300 rcf, 5 min, 4 ˚C). Nuclei were prepared by resuspending cells in 500 µl of ice-cold Nuclei Preparation Buffer (10 mM Tris-HCl pH 7.5 (Sigma #T2944-100ML), 10 mM NaCl (Sigma #S5150-1L), 3 mM MgCl2 (Ambion #AM9530G), 1% w/v BSA (Sigma #A8806-5), 1% v/v SUPERase-In RNase Inhibitor (20 U/µl, Thermo Fisher Scientific #AM2696), 0.1% v/v Tween-20 (Sigma #P7949-500ML), 0.1% v/v IGEPAL CA-630 (Sigma #I8896-50ML), 0.01% v/v Digitonin (Promega #G944A)), followed by 5 min of incubation on ice. Lysis of the plasma membrane was stopped by adding 5 ml of ice-cold Nuclei Wash Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor, 0.1% v/v Tween-20). Nuclei were collected by centrifugation (500 rcf, 5 min, 4 ˚C), resuspended in 200 µl of ice-cold PBS-BSA-SUPERase (1x PBS supplemented with 1% w/v BSA and 1% v/v SUPERase-In RNase Inhibi-tor) and filtered through a cell strainer (40 µM or 70 µM depending on the cell size). We then used 5 µl of the sample for cell counting in duplicates on a CASY device (Schärfe System) and diluted to 5,000 nuclei per µl with ice-cold PBS-BSA-SUPERase. We immediately proceeded with the reverse transcription step of scifi-RNA-seq. Methanol fixation of cells: A total of 5 million cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifuga-tion (300 rcf, 5 min, 4 ˚C) and fixed in 5 ml of ice-cold methanol (Fisher Scientific #M/4000/17) at -20 ˚C for 10 min. After two additional washes (centrifugation: 300 rcf, 5 min, 4 ˚C) with 5 ml of ice-cold PBS-BSA-SUPERase (1x PBS supplemented with 1% w/v BSA (Sigma #A8806-5) and 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific #AM2696)) permeabilized cells were resuspended in 200 µl of ice-cold PBS-BSA-SUPERase, and filtered through a cell strainer (40 µM or 70 µM depending on the cell size). We then used 5 µl of the sample for cell counting in duplicates on a CASY device (Schärfe System) and diluted to 5,000 cells per µl with ice-cold PBS-BSA-SUPERase. We immediately proceeded with the reverse transcription step of scifi-RNA-seq. Fixed nuclei suspension: A total of 5 million primary cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifugation (300 rcf, 5 min, 4 ˚C). Nuclei were prepared by resuspending cells in 500 µl of ice-cold Nu-clei Preparation Buffer without Digitonin and without Tween-20 (10 mM Tris-HCl pH 7.5 (Sigma #T2944-100ML), 10 mM NaCl (Sigma #S5150-1L), 3 mM MgCl2 (Ambion #AM9530G), 1% w/v BSA (Sigma #A8806-5), 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific #AM2696), 0.1% v/v IGEPAL CA-630 (Sigma #I8896-50ML)), followed by 5 min of incubation on ice. Lysis of the plasma membrane was stopped by addition of 5 ml of Nuclei Wash Buffer without Tween-20 (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor). Nuclei were collected by centrifugation (500 rcf, 5 min, 4 ˚C), and fixed in 5 ml of ice-cold 1x PBS containing 1% or 4% Formaldehyde (Thermo Fisher Scientific #28908) for 15 min on ice. Fixed nuclei were collected (500 rcf, 5 min, 4 ˚C), the pellet was resuspended in 1.5 ml of ice-cold Nuclei Wash Buffer without Tween-20 and transferred to a 1.5 ml tube. After one more wash with 1.5 ml of ice-cold Nuclei Wash Buffer without Tween-20 (500 rcf, 5 min, 4 ˚C), fixed nuclei were resuspended in 200 µl of Nuclei Wash Buffer without Tween-20, snap-frozen in liquid nitrogen and stored at -80 ˚C.  For scifi-RNA-seq, frozen samples were thawed in a 37 ˚C water bath for exactly 1 min, and immediately placed on ice. Following centrifugation (500 rcf, 5 min, 4 ˚C), fixed nuclei were resuspended in 250 µl of ice-cold Permeabilization Buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor, 0.01% v/v Digitonin (Promega #G944A), 0.1% v/v Tween-20 (Sigma #P7949-500ML)). After 5 min of incubation on ice, 250 µl of Nuclei Wash Buffer without Tween-20 were added per sample, and nuclei were collected (500 rcf, 5 min, 4 ˚C). After one more wash with 250 µl of Nuclei Wash Buffer without Tween-20, nuclei were taken up in 100 µl of 1x PBS containing 1% w/v BSA and 1% v/v SUPERase-In RNase Inhibitor. We used 5 µl for cell counting in duplicates on a CASY device (Schärfe Systems) and diluted to 5,000 nuclei per µl with PBS-BSA-SUPERase. We immediately proceeded with the reverse transcription step of scifi-RNA-seq." Reverse Transcription: 384 indexed reverse transcription primers were synthesized by Sigma Aldrich and obtained at 100 µM concentration in EB Buffer on 96-well plates (sequences are provided in Supplementary Table 1). 384-well plates with barcoded oligo-dT primers were prepared prior to the experiment and stored at -20 ˚C (1 µl of 25 µM per well). 10,000 permeabilized cells or nuclei (2 µl of a 5,000/µl suspension) were added to the pre-dispensed primers in each well, and the assignment of samples to wells was recorded. The plate was incubated for 5 min at 55 ˚C to resolve RNA secondary structures, then placed immediately on ice to prevent their re-formation. Per well, a mix of 3 µl nuclease-free water, 2 µl 5x Reverse Transcription Buffer, 0.5 µl of 100 mM DTT (freshly diluted from Sigma #646563-10x.5ML), 0.5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 0.5 µl of RNaseOUT RNase inhibitor (40 U/ml, Thermo Fisher Scientific #10777019), and 0.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753) were added. The reverse transcription was incubated as follows (with heated lid set to 60 ˚C): 50 ˚C for 10 min, 3 cycles of [8 ˚C for 12 s, 15 ˚C for 45 s, 20 ˚C for 45 s, 30 ˚C for 30 s, 42 ˚C for 2 min, 50 ˚C for 3 min], 50 ˚C for 5 min, store at 4 ˚C.  Cell/Nuclei recovery and pooling: Processed cells/nuclei were recovered from the 384-well plate and pooled in one 15 ml tube per plate on ice. Wells were washed with ice-cold 1x PBS containing 1% BSA, which was transferred to the same tube for maximum recovery. The volume was topped up to 15 ml with 1x PBS containing 1% BSA, and nuclei were collected (500 rcf, 5 min, 4 ˚C). The resulting pellet was resuspended in 1.0 ml of 1x Ampligase Reaction Buffer (Lucigen #A0102K), filtered through a cell strainer (40 µm or 70 µm depending on the cell/nuclei size) into a 1.5 ml tube, and centrifuged (500 rcf, 5 min, 4 ˚C). The supernatant was removed almost completely, and the tube was centrifuged briefly (500 rcf, 30 s, 4 ˚C) to collect the remaining liquid at the bottom of the tube. Typically, this resulted in ~10 µl of a highly concentrated suspension, which was diluted 1:200 with 1x Ampligase Buffer and counted in a Fuchs Rosenthal counting chamber (Incyto #DHC-F01). The desired number of cells/nuclei was brought to a volume of 15 µl with 1x Ampligase Reaction Buffer (Lucigen #A0102K).  Microfluidic thermoligation barcoding: Unused channels in the Chromium Chip E (10x Genomics #2000121) were filled with 75 µl (inlet 1), 40 µl (inlet 2), and 240 µl (inlet 3) of 50% glycerol solution (Sigma #G5516-100ML). Right before loading the chip, a mix of 47.4 µl nuclease-free water, 11.5 µl of 10x Ampligase Reaction Buffer (Lucigen #A0102K), 2.3 µl of 100 U/µl Ampligase (Lucigen #A0102K), 1.5 µl of Reducing Agent B (10x Genomics #2000087), and 2.3 µl of 100 µM Bridge Oligo (sequence provided in Supplementary Table 1) was added per sample. The microfluidic chip was loaded with 75 µl of cells or nuclei in thermoligation mix (inlet 1), 40 µl of Single Cell ATAC Gel Beads (inlet 2, 10x Genomics #2000132), and 240 µl of Partitioning Oil (inlet 3, 10x Genomics #220088) and run on the Chromium system. For thermoligation barcoding, the droplet emulsion was incubated as follows (heated lid set to 105 ˚C, volume set to 100 µl): 12 c