NextSeq 500 paired end sequencing; A flexible microfluidic system for single-cell transcriptome profiling elucidates phased transcriptional regulators of cell cycle
A flexible microfluidic system for single-cell transcriptome profiling elucidates phased transcriptional regulators of cell cycle
HEK293 or 3T3 cells were cultured in DMEM with 10% fetal bovine serum (Life Techologies) and 1× penicillin-streptomycin (Life Technologies). Cells were trypsinised for 5 minutes with TrypLE (Life Technologies) before being collected and spun down for 5 min at 300 g. The pellet was resuspended in 1 ml of PBS-BSA (1x PBS, 0.01% BSA) and spun again for 3 min at 300 g. The cells were resuspended in 1 ml of PBS, passed through a 40 μm cell strainer and counted. A concentration of 300 cells/μl in 250 μl of PBS- BSA was subsequently used to allow for the encapsulation of ~1 cell in every 20 droplets. For nuclei, nuclei isolation media (NIM) was prepared in advance (250mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris pH8) and pre-chilled. Cells were trypsinised for 5 minutes with TrypLE (Life Technologies) before being collected and spun down for 5 min at 300 g. The pellet was resuspended in 1 ml of PBS-BSA (1x PBS, 0.01% BSA, 0.02 U/μl supernasin) and spun again for 3 min at 300 g. The cells were resuspended in 1 ml of nuclei homogenisation buffer (NIM, 1 μM DTT, 1x Protease inhibitor, 0.1% Triton X-100, 0.04 U/μl RNasin, 0.02 U/μl Superasin) and mixed by gentle pipetting. Sample was then spun at 300g and 4oC for 5 minutes. Supernatant was discarded and the pellet was resuspended in 1 ml of PBS-BSA (0.01% BSA, 0.02 U/μl supernasin). Finally, sample was vortexed and filtered through a 40 μm strainer before nuclei quality was assessed with trypan blue and Hoechst staining and diluted to desired concentration for Nadia loading.    Cell or nuclei suspensions were captured using the Nadia system according to pre-programmed instrument protocols for drop-seq or sNuc-seq that were accessed through the instruments touch-screen interface. mRNA was captured on barcoded mRNA capture beads loaded onto Nadia platform mRNA bound beads were removed from the Nadia Instrument's collection chamber and transferred to a 50 ml falcon tube. Next, 30 mls of 6x SSC buffer (Life Technologies) and 1ml of 1H,1H,2H,2H-Perfluorooctan-1-ol (Sigma Aldrich) were added before mixing via inversion. After spinning at 1000g for 2 minutes the supernatant was removed and retained in a separate falcon whilst being careful not to disturb the beads at the oil-water interface. A further 30 mls of 6x SSC buffer were added to the original sample to disturb the beads before mixing via inversion. Oil was allowed to settle to the bottom before bead containing suspension was transferred to a new falcon tube. After disturbing the oil fraction with a 1 ml pipette to collect any missed beads, both falcons containing ~30 mls of bead containing SSC buffer were spun at 1000g and 4oC for 2 minutes. At this stage, ~26mls of supernatant was carefully removed from each tube whilst being careful not to disturb the beads. Beads were subsequently resuspended with retained buffer and transferred to a 1.5 ml eppendorf. Beads were spun down in a desktop micro-centrifuge and buffer removed. Additional bead fractions were added and the process repeated until all beads were collected. At this stage the buffer was removed and all beads washed by pipetting in 1 ml of 6x SSC buffer. Buffer was removed and beads were subsequently washed in 200 μl of 5x Maxima RT buffer (Life Technologies). Reverse transcription was performed in 200 μl of a 1x RT mix (80 μl nuclease free water, 40 μl of 5x Maxima RT buffer, 40 μl of 20% Ficoll PM-400, 20 μl of 10 mM dNTP mix, 5 μl of RNasin, 10 μl of Maxima H-RT enzyme, 5 μl of 100 μM TSO-RT primer) with the following conditions; 30 minutes at 23oC, 2 hours at 42oC. Throughout the process the sample was set to shake at 1100 rpm. Beads were subsequently spun down, RT mix removed and the beads washed in once in TE-SDS buffer (10 mM Tris pH 8, 1 mM EDTA, 0.5% SDS), twice in TE-TW buffer (10 mM Tris pH 8, 1 mM EDTA, 0.01% Tween-20) and once in 300 μl of 10 mM Tris pH 8. Beads were subsequently incubated for 45 minutes at 37oC and 1100 rpm in Exonuclease I mix (170 μl nuclease free water, 20 μl 10x Exonuclease I buffer, 10 μl Exonuclease I - Life Technologies). Beads were then washed once in TE-SDS buffer, twice in TE-TW buffer and then re-suspended in 300 μl of nuclease free water. Beads were subsequently counted with a haemocytometer after mixing 20 μl of beads with 20 μl of 20% PEG400 (Sigma Aldrich). An average of 4 counts were taken before test PCRs at different cycle numbers were performed with desired bead aliquots for each experiment (~2000-5000)  to gauge optimal cycles for final PCRs on subsequent beads. Specifically, PCR mix included 24.6 μl of nuclease free water, 0.4 μl of 100 μM TSO-PCR primer, and 25 μl of Kapa HiFi readymix (Roche Diagnostics). Cycling conditions were 95oC for 3 minutes, four cycles of 98oC for 20 seconds, 65oC for 45 seconds, 72oC for 3 minutes, followed by variable cycles (~9-14) of 98oC for 20 seconds, 67oC for 20 seconds, 72oC for 3 minutes. A final extension of 72oC for 3 minutes completed the PCR. At the end of elongation steps during the first four cycles, PCR tubes were removed from machine and beads suspended by gentle agitation. Following optimised PCRs of desired bead numbers, we enriched cDNA products longer than 300 base pairs using select-a-size spin columns (Zymogen) according to the manufacturer protocol. After bioanalyser evaluation and quantification of products, 550 pg of DNA was used as input for an Illumina Nextera tagmentation reaction according to manufacturer's protocol (15 μl Nextera PCR mastermix, 8 μl nuclease free water, 1 μl of 10 μM TSO-hybrid oligo, 1 μl of 10 μM Nextera N70X indexed oligo). This reaction reduced cDNA libraries to a size distribution suitable for Illumina sequencing, and added a common PCR handle for 12 cycles of final library amplification (95oC for 30 seconds, twelve cycles of 95oC for 10 seconds, 55oC for 30 seconds, 72oC for 30 seconds, final extension of 72oC for 3 minutes). Last, as shorter cDNA inserts are more likely to be overlap variable length poly-A tails, we again enriched for cDNA products longer than 300 base pairs using select-a-size spin columns (Zymogen) according to the manufacturer protocol. The final library profiles were then evaluated and quantified with a Bioanalyser, Qubit and Tapestation prior to sequencing.