4_GATCTG
Single-Cell RNA-Seq  in Physcomitrella patens
RNA and cell Isolation Physcomitrella patens tissue was cultured using standard methods according to Vidali et al., 200722. We used an NLS4 moss line expressing nuclear GFP for protoplasts fluorescence identification23. Total RNA was extracted via Quick-RNA MicroPrep (Zymoresearch) from 7 days old plant tissue. Plant tissue (about 300 mg) was frozen in liquid nitrogen, ground with a pre-chilled mortar and a pestle and homogenized in RNA lysis buffer. Single protoplasts were obtained as previously described24, briefly, plant tissue was digested with the enzyme cocktail Driselease (0.5%) (Sigma) dissolved in 8% mannitol. Using a spatula, 7 days old plant tissue was transferred into a petri dish containing 6 ml of Driselease solution, the petri dish was incubated at room temperature and gently shaken for 1 hour. The protoplast suspension was filtered through a 100 um mesh (BD Falcon 352350). Protoplasts were washed in an 8% mannitol solution, with 3 consecutive centrifugation and resuspension of protoplast pellet in fresh mannitol. The washed protoplasts, resuspended in 8% mannitol, were pipetted into a holed microscope slide (Plexiglas) with a microscope coverslip (glass) attached at the bottom with vacuum grease. The microscope slide was placed onto an inverted epifluorescence microscope Axiovert 200M (Zeiss). Protoplasts were extracted from the media with borosilicate glass capillaries (WPI, Inc), pulled with a flaming/brown micropipette puller, model-P-47 (Sutter Instrument). The glass capillary was connected to a manual pneumatic microinjector (CellTram Air Eppendorf). Single protoplasts were pipetted out of the mannitol suspension with the microinjector and pipetted into a PCR tube (VWR) containing 5 l of HBSS solution (Quality biological). The final volume was approximately 10 l. For detection of the protoplast in the tube, the cell was visualized by fluorescence GFP signal using a GFP filter cube GFP-30-LP-B-zhe zero (Semrock brightline). The tube was fast frozen in liquid nitrogen and stored at -80C until analysis. Tn5 enzyme purification Tn5 transposase enzyme was extracted according to Picelli et al., 2014 with a few modifications25. Briefly, the pTXB1-Tn5 vector (Addgene) was transformed into T7 lysIq competent cells (NEB). Single colonies were picked and two litre of bacterial culture in LB was grown at 37C; when the cultures reached OD 0.9, cells were induced with IPTG 0.25 mM and grown at 24C until OD 2.5. Cells were collected and resuspended in 100 ml HEGX buffer (10 mM Tris-HCl pH 7.5, 700 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, 1 Roche tablet, 1 mM PMSF (Sigma), and DNAse) and lysed with Microfluidizer cell disruptor (Microfluidics) at 80 PSI. Cells were pelleted for 30 minutes at 34000 g. The supernatant was incubated with chitin resin (NEB) in a conical tube for 2 hr at 4C to favour binding (batch binding), then loaded on a column. The column was 9/15 washed with 200 ml HEGX buffer, after which 12 ml of TGEX buffer with 100 mM DTT was loaded onto the column and 5.5 ml was drained out of the column. The column was left closed at 4C for 48 hr to ensure cleavage of the Tn5 from the intein. Elution was done in 1 ml aliquots and the concentration of the protein was measured via Bradford assay. The protein was concentrated with centrifugal filter units 10 K (Millipore). The activity of the protein was measured according to Picelli et al., 201425 . RNA preparation, reverse transcription and PCR For run 170420 and run 171108, total RNA was extracted from P. patens as described above and diluted to 2.5 pg/l (1 cell equivalent) or 25 pg/l (10 cell equivalent) in RNase free water and 0.56 l of Takara RNase Inibitor (Takara cat. 2313A) was added. The RNA was freeze-thawed three times at -80 C. For run 171108, individual protoplasts were harvested as described above in 5 l of Hanks Balanced Salt Solution (HBSS) and 0.56 l of Takara RNase Inibitor was added. The protoplasts were lysed by freeze-thawing three times at -80 C. The following tagmentation protocol was derived from Islam et al., 3 and was performed in sextuplicate using either purified P. patens RNA (technical replicates) or individual protoplast (biological replicates). For reverse transcription, 1 cell equivalent of RNA or protoplast lysate (21 pg of RNA) or 10 cell equivalents (210 pg of RNA) was mixed with template switching oligo, TSO3 (Supplementary Table S 1) that contained 10 nucleotide UMIs. The mixtures were incubated in a thermocycler at 72C for three minutes and then slow ramped to 42C at 0.1C/second. Once the mixture reached 42C, the temperature was held for two minutes. During the slow ramp incubation, an RT master mix (1X SuperScript II First Strand Buffer (Thermo Scientific 18064014), 3 mM MgCl2 (Thermo Scientific AB0359), 1.5 mM each of dNTP mix (Invitrogen 18427013), 4 mM DTT (Invitrogen 18064014), 18 U/l SuperScript II reverse transcriptase (Invitrogen 18064014), and 4 M FSP primer (Supplementary Table S 1)) was made. After the RNA/TSO3 mixture was incubated for two minutes at 42C, the RT master mix was also placed on the thermocycler and incubated at 42C for an additional minute. Next, RT master mix was added to each of the six replicate RNA/TSO3 mixtures. The RT reactions were incubated at 42C for 90 minutes, followed by 70C for 10 minutes to inactivate the RT enzyme. The RT reactions were stored overnight at -20C. The following day, half of the cDNA for each replicate was used for PCR (1X Advantage 2 PCR buffer (Takara 639201), dNTP mix 0.44 mM each (Invitrogen 18427013), 0.52 M MODIPCR primer (Supplementary Table S 1), 2X Advantage 2 polymerase mix (Takara 639201)). The PCR reactions were placed in a thermocycler and run with the following cycling parameters: Step 1: 95C for two minutes, Step 2: 98C for 30 seconds, Step 3: 60C for 20 seconds, Step 4: 70C for four minutes, Step 5: Repeat Steps 2-4 for 28 cycles, Step 6: 70C for five minutes, and Step 7: hold at 4C. During Step 4, at cycles 21, 23, 25, 27, and 29, 5 l aliquots were collected and placed on ice. The aliquots were placed back on the thermocycler at Step 6 for a final extension step. The PCR aliquots for each replicate were run in a 1% TAE agarose gel. The gel was stained with ethidium bromide/H2O solution and imaged on a Bio-rad Gel Doc XR+ imaging system. From the gel images, 27 cylces was determined to be the optimal number of PCR cycles for purified RNA and protoplasts. Following PCR optimization, the remaining cDNA for each replicate was used to repeat the PCR reactions described above. The cycling parameters were also repeated as described above but with 27 PCR cycles and without collecting aliquots. The PCR reactions were cleaned using 0.7X volumes of AmPure XP beads (Beckman Coulter A63881) and washed twice with 80% ethanol. The beads were incubated at room temperature for 7-15 minutes until they were completely dry. The PCR products were eluted in 14 l of EB buffer (Qiagen 19086). 12 l of eluted PCR products were collected. 1 l of PCR product for each replicate was quantified using a Qubit DNA High Sensitivity Assay Kit (ThermoFisher Scientific Q32851) and the Qubit3.0 fluorometer (ThermoFisher Scientific Q33216), as specified by the manufacturer. To assess the size distribution of the libraries , 1 l of each library was run on a 2100 Bioanalyzer (Agilent G2939A) using a High Sensitivity DNA Analysis kit (Agilent 5067-4626), as specified by the manufacturers instructions. The size distribution of the libraries ranged from 200-1000 bps. The libraries were stored overnight at -20C. Tagmentation and library amplification The following day, the PCR product was simultaneously fragmented and barcoded using Tn5 DNA transposase to transfer Illumina adaptors to the target DNA. For each replicate, PCR product was mixed with tagmentation buffer (50mM TAPS-NaOH pH 8.5 (Sigma T130-25G), 25mM MgCl2 (Ambion Am9530G), and 40% PEG 8000 (Sigma 83271-500ml-F)), 1X Tn5 assembly (10X Tn5 assembly Stock- 6.25 M barcoded adaptor, dialysis buffer (100 mM HEPES-KOH, pH 7.2, 0.2 mM NaCl, 0.2 mM EDTA, 2mM DTT, 20% glycerol), 40% glycerol, and 6.25 M Tn5 transposase). Each replicate received a Tn5 assembly containing a uniquely barcoded adapter. The reactions were incubated in a thermocycler at 55C for seven minutes and then transferred to ice. Proteinase K (0.5 g/l (Ambion AM2546)) was added to each reaction. The samples were incubated at 55C for seven minutes to inactivate the Tn5 transposase. Dynabeads M-280 Streptavidin beads (Invitrogen 11206D) were washed once with BWT (10 mM Tris-HCl , pH 7.5 (Invitrogen 15567-027), 1 mM EDTA (Ambion AM9260G), 2 M NaCl (Ambion AM9760G), 0.02% Tween-20 (Sigma 10/15 P9416-50ML)) and were resuspended in BWT. Washed Dynabeads M-280 Streptavidin beads were added to each tagmentation reaction and the mixture was rotated at room temperature for 10 minutes to allow the tagmentation libraries to bind to the beads. The bead slurries were placed on a DyaMag-96 side magnet for five minutes until the supernatant cleared. The supernatant was discarded and the beads were resuspended in PB buffer (Qiagen 19066). The bead slurry was placed back on a DyaMag-96 side magnet until the supernatant cleared. The supernatant was discarded and the beads were washed twice with TNT buffer (20 mM Tris-HCl , pH 7.5 (Invitrogen 15567-027), 50 mM NaCl (Ambion AM9760G), 0.02% Tween-20 (Sigma P9416-50ML)). The beads were resuspended in restriction mix (1X NEB Buffer 4 (New England Biolabs B7004S) and 0.4 U/l PvuI-HF (New England Biolabs R3150S)) to digest and remove unwanted 3 0 fragments carrying the PvuI recognition site. The bead slurry was place in a thermocycler and incubated at 37C for one hour. After incubation, the bead slurry was placed on a DyaMag-96 side magnet until the supernatant cleared. The supernatant was discarded and the beads were washed three times with TNT, as described above. After the third TNT wash, the beads were resuspended in EB buffer. Next, the tagmentation libraries were amplified by PCR to obtain enough material for Illumina sequencing. Each tagmentation library was mixed with 1X Advantage 2 PCR buffer, dNTP mix 0.2 mM each, 0.1 M P5 primer (Supplementary Table S 1), 0.1 M P7 primer (Supplementary Table S 1) and 1X Advantage 2 polymerase mix. The PCR reactions were placed in a thermocycler and run with the following cycling parameters: Step 1: 95C for two minutes, Step 2: 98C for 30 seconds, Step 3: 60C for 20 seconds, Step 4: 70C for four minutes, Step 5: Repeat Steps 2-4 for 9 cycles, Step 6: 70C for five minutes, and Step 7: hold at 4C overnight. The following morning, the amplified libraries were cleaned with 0.7X volumes of AmPure XP beads and washed twice with 80% ethanol. The residual ethanol was removed and the beads were incubated at room temperature for 7-15 minutes until they were completely dry. The amplified libraries were eluted in 14 l of EB buffer and 12 l of library was collected. The libraries were quantified using a Qubit DNA High Sensitivity Assay Kit and the Qubit3.0 fluorometer. The library size distribution was assessed using the 2100 Bioanalyzer and a High Sensitivity DNA Analysis kit. The size distribution of the libraries ranged from 200-700 bps. The libraries were stored at -20C until used for sequencing. Illumina Sequencing Each library was quantified by 2100 Bioanalyzer, as described above. The libraries were normalized to 10 nM, pooled in equimolar amounts and denatured as described by Illuminas Denature and Dilute Libraries Guide (Document 15050107 v03). The pools were clustered on a flowcell at 10 pM and sequenced on an Illumina HiSeq 2000 instrument using a TruSeq SBS Kitv3 (Illumina FC-401-3002, 50 cycles). MODR1P (Supplementary Table S 1) was used as the read 1 primer. For run 171108, MODRIP (Supplementary Table S 1) was used as the index read primer. 50 bp reads were generated for both runs along with 6 bp index reads for run 171108 corresponding to the RNA/protoplast-specific barcode
4_GATCTG
SUB4131845
4_GATCTG