Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: 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. 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 cycles of [98 ˚C for 30 s, 59 ˚C for 2 min], storage at 15 ˚C. The emulsion was broken by addition of 125 µl Recovery Agent (10x Genomics #220016), and 125 µl of the pink oil phase were removed by pipetting. The remaining sample was mixed with 200 µl of Dynabead Cleanup Master Mix (per reaction: 182 µl Cleanup Buffer (10x Genomics #2000088), 8 µl Dynabeads MyOne Silane (Thermo Fisher Scientific #37002D), 5 µl Reducing Agent B (10x Genomics #2000087), 5 µl of nuclease-free water). After 10 min of incubation at room temperature, samples were washed twice with 200 µl of freshly prepared 80% ethanol (Merck #603-002-00-5) and eluted in 40.5 µl of EB Buffer (Qiagen #19086) containing 0.1% Tween (Sigma #P7949-500ML) and 1% v/v Reducing Agent B. Bead clumps were sheared with a 10 µl pipette or needle. 40 µl of the sample were transferred to a fresh tube strip and subjected to a 1.0x cleanup with SPRIselect beads (Beckman Coulter #B23318), eluting in 22 µl of EB Buffer. Template switching: 20 µl of sample from the previous step were mixed with 10 µl of 5x Reverse Transcription Buffer, 10 µl of Ficoll PM-400 (20%, Sigma #F5415-50ML), 5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 1.25 µl of Recombinant Ribonuclease Inhibitor (Takara #2313A), 1.25 µl of 100 µM Template Switching Oligo (sequence provided in Supplementary Table 1), and 2.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753). The template switching reaction was incubated for 30 min at 25 ˚C, 90 min at 42 ˚C, storage at 4 ˚C, and cleaned with a 1.0x SPRI cleanup, eluting in 17 µl of EB buffer. cDNA enrichment: 15 µl of the above sample were mixed with 33 µl of nuclease-free water, 50 µl of NEBNext High-Fidelity 2x PCR Master Mix (NEB #M0541S), 0.5 µl of 100 µM Partial P5 primer, 0.5 µl of 100 µM TSO Enrichment Primer (sequences provided in Supplementary Table 1), and 1 µl of 100x SYBR Green in DMSO (Life Technologies #S7563). cDNA was amplified in a thermocycler as follows: 98 ˚C for 30 s, cycle until fluorescent signal >1000 RFU [98 ˚C for 20 s, 65 ˚C for 30 s, 72 ˚C for 3 min], 72 ˚C for 5 min in another thermocycler, storage at 4 ˚C. cDNA was cleaned by one 0.8x SPRI cleanup followed by a 0.6x SPRI cleanup, quantified with a Qubit HS assay (ThermoFisher Scientific #Q32854), and 1.5 ng were checked on a Bioanalyzer High-Sensitivity DNA chip (Agilent #5067-4626 and 5067-4627). Tagmentation: To achieve maximum library complexity, the entire sample was processed in multiple tagmentation reactions with 1 ng input each. cDNA was diluted to 0.2 ng/µl with nuclease-free water and 5 µl (1 ng) per reaction were distributed into a 96-well plate on ice. A mix of 11.25 µl nuclease-free water, 5 µl of 5x Tn5 Reaction Buffer (50 mM TAPS (Sigma #T9659-100G), 25 mM MgCl2 (Ambion #AM9530G), pH adjusted to 8.5, sterile-filtered), 2.5 µl of Dimethylformamide (Sigma #D4551-250ML), and 1.25 µl of freshly diluted i7-only transposome (prepared as described above and diluted 1:4.5 in Tn5 Dilution Buffer (50 mM Tris-HCl pH 7.5 (Sigma #T2944-100ML), 100 mM NaCl (Sigma #S5150-1L), 0.1 mM EDTA (Invitrogen #AM9260G), 50% glycerol (Sigma #G5516-100ML), 0.1% Triton-X100 (Sigma