title: NextSeq 500 paired end sequencing; Transcriptomic characterization of the human cell cycle in individual unsynchronized cells
design: Transcriptomic characterization of the human cell cycle in individual unsynchronized cells
library_construction_protocol: For single-cell sampling, 1 million trypsinized MLS 1765-92 cells/ml were resuspended in Hanks' balanced salt solution (Thermo Fisher Scientific) and stained for nuclear DNA using Vybrant DyeCycle Violet stain (Thermo Fisher Scientific) at 37°C and a final concentration of 5 µM for 30 min. After staining, the cells were resuspended in Hanks' balanced salt solution and filtered via a 70 µm cell strainer (BD Biosciences) to remove cell aggregates. The cells were kept on ice until sorting. Single cells were sorted according to cell cycle phases G1, S, and G2/M into 96-well plates (Thermo Fisher Scientific) containing 5 µl 1 µg/µl BSA in 2.5% glycerol (Thermo Fisher Scientific) and 0.2% Triton X-100 (Sigma-Aldrich) in DNase/RNase-free water (Thermo Fisher Scientific) using a BD FACSAria II (Becton Dickinson) instrument and the FACSDiva software. Collected cells were frozen on dry ice immediately after sorting and stored at -80°C. MLS 1765-92 cells were cultured at 37°C and 5% CO2 in RPMI 1640 GlutaMax medium supplemented with 5% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were passaged with 0.25% trypsin containing 0.5 mM EDTA (all Thermo Fisher Scientific). To generate sequencing-ready libraries, the Smart-seq2 protocol was used, with minor changes. In short, 1 µM adapter sequence-containing oligo-dT (5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3', Sigma-Aldrich), 1 mM dNTP (Sigma-Aldrich) and ERCC spike-in controls (corresponding to 1 µl of 1:2,500,000 diluted stock solution; Thermo Fisher Scientific) were incubated with direct lysed cells in a volume of 6.7 µl at 72˚C for 3 min. Reverse transcription (RT) was then performed in 15 µl by adding 1x first-strand buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, and 3 mM MgCl2, Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 5 mM DTT, additional 10 mM MgCl2 (both Thermo Fisher Scientific), 0.6 µM adapter sequence-containing template switching oligonucleotide (5'-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3' where rG = riboguanosine and +G = locked nucleic acid modified guanosine, Eurogentec), 15 U RNaseOUT and 150 U SuperScript II enzyme (both Thermo Fisher Scientific) RT was performed at 42°C for 90 min and 70°C for 15 min before being chilled to 4°C. Final RT concentrations are shown. cDNA was stored at -20°C until further use. Preamplification of 7.5 µl cDNA was then performed in 50 µl, containing 1x KAPA Hifi HotStart Ready Mix (KAPA Biosystems) and 60 nM adapter sequence-matching primer (5'-AAGCAGTGGTATCAACGCAGAGT-3', Sigma-Aldrich), as follows: denaturation at 98°C for 3 min followed by 24 cycles of amplification (98°C for 20 s, 67°C for 15 s, and 72°C for 6 min) and a final incubation at 72°C for 5 min before being chilled to 4°C. Preamplified cDNA was purified using the AMPure XP beads kit (Agencourt, Beckman Coulter), according to the Smart-seq2 protocol, with a sample to beads volume ratio of 0.8. Purified cDNA was eluted in 15 µl water and stored at -20°C until further use. Concentration, fragment length distribution, and total transcriptome amount of preamplified libraries were analyzed by capillary gel electrophoresis using the High Sensitivity DNA Kit (Agilent) on a 2100 Bioanalyzer (Agilent). Sequencing libraries were generated using the Nextera XT DNA Sample Preparation Kit and Nextera XT v2 Index Kit (both Illumina), following the manufacturer's recommendations with minor changes. In short, 0.1 ng of purified and preamplified cDNA were tagmented in 20 µl, containing 10 µl TD buffer and 5 µl ATM at 55°C for 5 min. Tagmentation was stopped by adding 5 µl of NT buffer at room temperature incubating for another 5 min. Incorporation of index sequences was performed in 50 µl, containing 15 µl NPM PCR master mix solution, 5 µl i5 index primer and 5 µl i7 index primer, as follows: heating to 72°C for 3 min, denaturation at 95°C for 30 s and 16 cycles of amplification (95°C for 10 s, 55°C for 30 s, and 72°C for 30 s), and a final extension step at 72°C for 5 min before chilling to 10°C. Indexed samples were purified using the AMPure XP beads, as described above, with a sample to beads volume ratio of 0.6. Mean fragment length and concentration of indexed single-cell libraries were assessed using High Sensitivity DNA Kit (Agilent) and dsDNA High Sensitivity Assay Kit (Qubit, Thermo Fisher Scientific), respectively. Single-cell libraries were diluted to 2 nM, pooled and sequenced by the Genomics Core facility at the University of Gothenburg.