GSM8015424: NIH-3T3 and K562 RNA (without blocking oligo); Homo sapiens; Mus musculus; RNA-Seq
mouse NIH-3T3 cells were washed once with PBS and dissociated with Accutase (Stem Cell Technologies, catalog no. 07920). Two million NIH-3T3 and K562 cells were collected and fixed in a 3% glyoxal solution (40% glyoxal (Merck, catalog no. 128465) 0.75% acetic acid; 112.5 mM NaOH to adjust the solution to pH5.0) for 7 min at room temperature. For cryopreservation, cells were washed after fixation with RSB-1%BSA-RI (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM DTT, 1% BSA, in-house made RNasin (referred to RNasin hereafter)) and slowly frozen (Freezing buffer: 50 mM Tris-HCl pH 7.5, 5 mM MgAc, 0.1 mM EDTA and 25% glycerol) (cryopreserved cells indicated in sampleindexSheet.csv). On the day of pre-indexing, fresh cells were processed as described above, while cryopreserved cells were thawed slowly on ice and washed with RSB-1%BSA-RI. Nuclei were extracted with 1x lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween20, RNasin, 1 mM DTT, 1% BSA, 0.025% IGEPAL CA-630 and 0.01% Digitonin) incubating samples for 5 min on ice. Next, nuclei were washed with wash buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween20, RNasin, 1 mM DTT, 1% BSA) and filtered through a 40 µm cell strainer (Falcon, catalog no. 352340). Transposome assembly: For accessible DNA transposition transposome assembly, 100 µM of oligonucleotides with the Nextera Read1 sequence and 100 µM of oligonucleotides with a Read2-sample_index-spacer structure were annealed with 100 µM mosaic end-complement oligonucleotides with a 3' dideoxynucleotide end (ddc) at a 1:1:2 ratio by heating for 95 °C for 3 minutes followed by cooling down to 25 °C at a ramp rate of -1 °C/min (oligonucleotide sequences provided in supplementary table 1). Annealed oligos were mixed with an equal volume of 100% glycerol and stored at -20 °C until use. Finally, the annealed oligos were diluted 1:1 with H2O and mixed at equal volume with in-house produced Tn5 (at 1 mg/ml in 50 mM Tris, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% NP-40, and 50% glycerol, production details in methods), and incubated for 30-60 min at room temperature. For cDNA/RNA tagmentation transposome preparation, 100 µM of oligonucleotides with a Nextera Read1 sequence overhang were annealed with 100 µM of the Tn5-mosaic end oligonucleotides at a 1:1 (v/v) ratio as described above. Annealed oligos were mixed with an equal volume of 100% glycerol and stored at -20 °C until use. For transposome assembly, annealed oligos were mixed with the in-house produced Tn5 at a 1:1 ratio for 30 min at RT. Assembled Tn5 was stored at -20 °C until use.  Accessible DNA transposition:Isolated and fixed nuclei were resuspended in a transposition mix containing 38.8 mM Tris-acetate, 77.6 mM potassium acetate, 11.8 mM magnesium acetate, and 18.8 % dimethylformamide supplemented with 0.005x protease inhibitor cocktail (Roche, catalog no. 11697498001), 0.4 U/µl SUPERaseIn (Thermo Fisher, catalog no. AM2694), 1.2 U/µl RiboLock (Thermo Fisher, catalog no. EO0382) and the barcoded Tn5 transposomes (1:10 (v/v); 938 µM) and incubated at 30 °C for 30 min with shaking at 400 rpm. The reaction was terminated by adding an equal volume of 2x stop buffer (10 mM Tris-HCl pH 7.5, 20 mM EDTA pH 8.0, 2% BSA).   Reverse transcription: Transposed nuclei were washed 3 times with RSB-1%BSA-RI and resuspended in reverse transcription mix containing 1x RT buffer (50 mM Tris-HCl pH 8.0, 75 mM NaCl, 3 mM MgCl2, 10 mM DTT), 0.5 mM dNTPs, 1 U/µl Protector RNase inhibitor (Roche, catalog no. 3335402001), 0.2 U/µl SUPERaseIn (Thermo Fisher, catalog no. AM2694), 10 U/µl Maxima H Minus RT (Thermo Fisher catalog no. EP0752) and 5 µM of the respective barcoded oligo-dT primer (oligonucleotide sequences provided in supplementary table 1). For a subset of samples 12% PEG 8000 (w/v; Jena Bioscience, CSS-256) was included in the reverse transcription reaction (indicated in sampleIndexSheet.csv). The samples were incubated at 50 °C for 10 min, followed by 3 thermal cycles (8 °C for 12 sec, 15 °C for 45 sec, 20 °C for 45 sec, 30 °C for 30 sec, 42 °C for 2 min and 50 °C for 3 min), and finally incubated at 50 °C for 5 min.   cDNA/RNA tagmentation: Following the RT reaction, all samples were pooled in RSB-RI and washed twice with RSB-1%BSA-RI. The collected nuclei pool was counted, and the following reaction volume was adjusted according to the total number of nuclei (100 µl reaction for each 100,000 nuclei). Nuclei were resuspended in the transposition mix containing 1x transposition buffer (38.8 mM Tris-acetate, 77.6 mM potassium acetate, 11.8 mM magnesium acetate, and 18.8% dimethylformamide and 0.005x protease inhibitor cocktail and a 1:100 ratio (v/v) of assembled Tn5 (93.8 µM) The tagmentation was performed on a shaking thermoblock at 37 °C for 30 min at 400 rpm. The reaction was terminated by adding an equal volume of 2x stop buffer.   Gap filling: Next, the sample pool was washed twice with RSB-1%BSA. Sample pool was resuspended a mix containing 1x RT buffer (50 mM Tris-HCl pH 8.0, 75 mM NaCl, 3 mM MgCl2, 10 mM DTT), 0.5 mM dNTPs, 8 U/µl Maxima H Minus RT (Thermo Fisher catalog no. EP0752) and 2 U/µl Thermolabile Exonuclease I (New England Biolabs, catalog no. M0568S) and incubated for 15 min at 37 °C on a shaking thermoblock at 400 rpm. Afterwards the sample was washed twice with RSB-1%BSA filtered through a 40 µm cell strainer and count.   Cell barcoding: The sample was cleaned prior loading of the 10x Genomics chip using a glycerol-based buffer (50 mM Tris-HCl pH 7.5, 5 mM Mg-acetate, 0.1 mM EDTA, 25% glycerol). For this, nuclei were resuspended in a glycerol-based buffer, layered on top of an additional 500 µl glycerol-based buffer and centrifuged at 800 x g for 15 minutes. The supernatant was discarded, and the pellet was washed twice with RSB-1%BSA and filtered through a 40 µM Flowmi cell strainer (Sigma-Aldrich, catalog no. BAH136800040). The nuclei were counted and loaded into the Chromium Controller (10x Genomics) according to 10x Genomics single-cell-ATAC standard protocol with minor adaptations. In brief, nuclei in 1x Nuclei buffer (10x Genomics) were mixed with barcoding reagents (Barcoding Reagent B, Reducing Reagent B and Barcoding Enzyme). The ”with blocking oligo” libraries were supplemented with ~500 nM of a 3'inverted-dT blocking oligonucleotide (oligonucleotide sequence provided in supplementary table 1). For Chip loading, the standard 10x workflow was followed. In brief, 70 µl of the sample, 50 µl of Barcoding Gel Beads, 40 µl Partitioning Oil were loaded into the respective channels. After the Chromium run, the droplet emulsion was transferred to 10x verified PCR tubes (Eppendorf, catalog no. EP0030124359) and the cell barcoding reaction was run in the thermocycler as follows: 5 min at 72 °C, 30 seconds at 98 °C, 4 or 12 cycles (98 °C 10 seconds, 59 °C 30 seconds and 72 °C 1 min) (cycle amount indicated in sampleindexSheet.csv). In the ”with blocking oligo” library, only 20% of the droplet emulsion was further processed to limit the sequencing requirements. Sample cleanup was performed following the standard 10x Genomics protocol with minor modifications: 125 µl Recovery Reagent was added to sample and inverted 10-15 times. After brief centrifugation, 125 µl of the pink oil phase was discarded and the aqueous phase was mixed with a dynabead cleanup mastermix (182 µl Cleanup Buffer, 13 µl Dynabeads MyOne SILANE and 5 µl Reducing Agent B (10x Genomics)) and incubated for 10 min at RT. Samples were placed on a magnetic separator and washed 2 times with 80% ethanol before eluting them in 42 µl of elution buffer. Finally, samples were subjected to a 1.4x SPRI cleanup (Beckman Coulter, catalog no. B23318).   Pre- and final library amplification: Following GEM generation, cell barcoding and cleanup, samples containing a mixture of single-cell ATAC and RNA libraries were resuspended in the pre-amplification mastermix containing 1x NEBNext HF 2x PCR Master Mix (NEB, catalog no. M0541L) and a combination of three primers: partial-P5-PTO primer (common for both modalities), TruseqR2 (specific for the RNA modality) and an ATAC-spacer primer (specific for ATAC modality) each at a final concentration of 500 nM (oligonucleotide sequences provided in supplementary table 1). Samples were incubated in the thermal cycle as follows: 98 °C 1 mi, 5 cycles of 98 °C for 20 sec, 63 °C for 20 sec and 72 °C for 20 sec), 72 °C for 1 min and then subjected to a 1.2x SPRI cleanup eluting in 42 µl EB buffer (Qiagen). Each sample was split into two fractions and each of them was further amplified with modality-specific primers: a partial-P5-PTO/P7-index-spacer primer pair for the ATAC libraries and a partial-P5-PTO/P7-index-TruseqR2 primer pair for the gene expression libraries (oligonucleotide sequences provided in supplementary table 1). The PCR programme for both library types was as follows: 98 °C for 1 min, 5 cycles of 98 °C for 20 sec, 63 °C for 20 sec and 72 °C for 20 sec followed by a final extension step at 72 °C for 1 min. To estimate the number of endpoint PCR cycles required for each library, 1/20th of the total library volume was used to perform a qPCR reaction using library-specific primers, and the number of cycles required to reach 50% of saturation was calculated. Finalized ATAC libraries were subjected to a 1.2x SPRI cleanup and RNA libraries to 0.72x SPRI cleanup. The concentration of the libraries was measured using Qubit 1x dsDNA-HS (Thermo Scientific, catalog no. Q33230) and the library fragment distribution was assessed using Bioanalyzer High Sensitivity DNA kit (Agilent, catalog no. 5067-4626).