GSM5710513: Fixed_D3_1589_mRNA; Homo sapiens; RNA-Seq
Day 11 cells were harvested, counted and split in two tubes, one to fixed/permeabilize and one for live cells analysis. Fixed/permeabilized cells were immediately fixed by adding 2x concentrated fixative to the cell suspension (5 mM dithiobis (succinimidyl propionate) (DSP) (Thermo Fisher Scientific, USA) and 5 mM succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Thermo Fisher Scientific, USA) in PBS (Thermo Fisher Scientific, USA). Cells were incubated at room temperature for 45 minutes while gently shaking. Fixed cells were quenched and permeabilized with 100 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.1% Triton X100 (Thermo Fisher Scientific, USA) for 10 minutes at room temperature. After permeabilization, cells were washed once and blocked for 45 minutes in 0.5X protein-free blocking buffer (Thermo Fisher Scientific, USA) with 0.2 mg/ml dextran sulfate (Sigma Aldrich, USA) and 0.5 U/ml RNasin plus (Promega, USA) in PBS (Thermo Fisher Scientific, USA). Cells were stained in a blocking buffer containing DNA-tagged antibodies (Supplementary Table 2) and PE-Cy7 conjugated CD38 Ab (Supplementary Table 1) for one hour at room temperature. Following staining, cells were washed twice with the blocking buffer and re-suspended in PBS containing 5 mg/ml BSA (Thermo Fisher Scientific, USA) and 0.5 U/ml RNsing plus (Promega, USA) and 0.1μg/ml DAPI (Biolegend, USA). Non-fixed cells were processed immediately and were washed twice in ice cold blocking buffer. Cells were stained in ice cold blocking buffer containing DNA-tagged Abs (Supplementary Table 3) and PE-Cy7 conjugated CD38 Ab (Supplementary Table 1). Cells were incubated on ice in the dark for 20min. Cells were washed three times in ice cold blocking buffer and re-suspended in blocking buffer containing 0.1μg/ml DAPI (Biolegend, USA). Cells were sorted single-cell in 384-wells PCR plates (Biorad, USA) using the FACS Melody (BD Biosciences, USA). Cells were sorted in 100nl water containing 7.5 ng/μl unique Cellseq-2 primers Supplementary Table 4) and 5 μl miniral oil (Sigma Aldrich, USA). Plates were stored at -80 °C until further use. Plates were thawed on ice before further processing. Reverse crosslinking was performed by dispensing 50 nl of a 3X reverse-crosslinking buffer [6 mM dNTP, 150 mM Tris pH 8, 90 mM DTT, 0.1% Triton X100, 6 U/μl RNAsin Plus (Promega)] into the wells and incubating 45 minutes at 25 °C. Hereafter, the reactions were incubated 5 minutes at 65 °C and cooled to 4 °C. Next, reverse transcription was performed in a total reaction volume of 250 nl using the Maxima H minus Reverse transcriptase (Thermo Scientific). The samples were heated to 85 °C for 5 minutes and the slowly cooled down by decreasing the temperature 5 degrees every 30 seconds until reaching 10 °C. Next, the samples were pooled and purified. During purification using 0.6x AMPure XP machnetic beads (Beckman Coulter, USA, the smaller antibody barcodes were separated from the large mRNAs by size selection. After the larger mRNAs bound to the beads the supernatant containing the Ab barcodes was taken and prepared separately. For the mRNA part, second strand synthesis and in vitro transcription were performed as described. Reverse transcription of the amplified RNA was performed with Maxima H minus Reverse Transcriptase (Thermo Scientific) using a combination of a random octamer primer. Library preparation PCR was performed in two steps. First a library pre-amplification with short primers was performed using the Herculase II enzyme (Agilent) to minimize amplification bias. Next, adapter extension was performed using a PCR reaction with Herculase II (Agilent) and the following oligonucleotides , Reverse indexing oligonucleotide.  Protein library preparation After size separation of the protein and mRNA libraries, the protein library was purified with 1.5x AMPure XP magnetic beads and eluted in 10 µL of nuclease-free water. Subsequently, the sample was PCR amplified after prior determination of the number of cycles needed by qPCR: 0.5 µL of purified material was mixed with 6.5 µL nuclease-free water, 10 µL of 2x Kapa HiFi HotStart PCR mix (Roche, Switzerland), 1 µL of 20X EvaGreen Dye (Thermo Fisher Scientific, USA), and 2 µL of REV unique indexing primer and the Nextera primer, primer mix (5 µM each) (Biolegio, The Netherlands). Then the PCR reaction was started by adding: 0.5 µL of nuclease-free water, 12.5 µL of 2X Kapa HiFi HotStart PCR mix (Roche, Switzerland), and 2.5 µL of REV unique indexing primer and the Nextera primer mix (5 µM each) (Biolegio, The Netherlands) to the 9.5 µL of the sample. The PCR thermal cycling was as follows: 2 minutes at 95 °C, followed by x cycles of (x = number previously determined by qPCR): 30 seconds at 95 °C, 30 seconds at 64 °C and 40 seconds at 72 °C, and terminated with 5 minute-incubation at 72 °C. PCR amplified product was purified by addition of 1.2X AMPure XP magnetic beads, eluted in 10 µl water and its quality was verified by ds-DNA concentration measurement with Qubit ds-DNA High Sensitivity assay (Thermo Fisher Scientific, USA) and Bioanalyzer (Agilent 2100, USA) characterization. If the quality of the samples was high enough, the protein libraries were sequenced together with the mRNA libraries on with NextSeq500 (targeting 50 million reads per sample).
GEO Accession
GSM5710513