Single-nucleus total RNA-sequencing of C2C12 myoblasts via in situ polyadenylation

Current methods for single-nucleus RNA-sequencing (snRNAseq) are limited to assaying endogenously polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNAs enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based single-nucleus transcriptomics to the total transcriptome. We apply standard snRNAseq and total snRNAseq to C2C12 myoblasts. Our in situ polyadenylation strategy relies on a brief, low-cost add-on to a widely used protocol for single-cell/single-nucleus RNA-sequencing, and thus could be broadly and quickly adopted. Single-nucleus RNA-sequencing of the total transcriptome will enable new insights into gene regulation and biology. Single-nucleus total RNA-sequencing was performed using a modified version of the Chromium protocol. C2C12 cells were grown to 90% confluence and collected with 0.25% TrypLE (Thermo Fisher Scientific). Nuclei were isolated similar to Petrany et al, Nature Communications, 2020. Cells were pelleted by centrifugation at 500 x g, at 4ºC, for 5 minutes, and resuspended in 6 ml of chilled homogenization buffer (0.25M sucrose, 1% bovine serum albumin, 1X phosphate-buffered saline, 0.2U/µl SUPERase•In RNase Inhibitor (Thermo Fisher Scientific #AM2694), nuclease-free H2O). 1ml of chilled 2.5% Triton-X100 diluted in 1X phosphate-buffered saline was then added. Cells were then incubated on ice for 5 minutes, then pelleted by centrifugation at 1000 x g, at 4ºC, for 5 minutes. Nuclei were then resuspended in 1X phosphate-buffered saline and counted using trypan blue. Five million nuclei were suspended in 200ul of 1X phosphate-buffered saline, then 800ul of ice-cold methanol was added dropwise to fix. Nuclei were then stored at -20ºC overnight. On the day of the experiment, nuclei were removed from -20ºC and incubated on ice for five minutes. Nuclei were then pelleted by centrifugation at 1000 x g, at 4ºC, for 5 minutes and resuspended in 200 µl wash resuspension buffer (0.04% bovine serum albumin, 1mM DTT, 0.2U/µl Protector RNase Inhibitor (Roche #3335402001), 3X SSC buffer (Thermo Fisher Scientific #15557044 ), nuclease-free H2O). Nuclei were then pelleted by centrifugation at 1000 x g, at 4ºC, for 5 minutes and washed in 200µl of 1X wash buffer (40µl 5X yPAP Reaction Buffer, 4µl 40U/µl Protector RNase Inhibitor, 156µl nuclease-free H2O), 1.6U/µl Protector RNase Inhibitor (Roche #3335402001), nuclease-free H2O). In situ polyadenylation was then performed by suspending nuclei in 50µl of yPAP enzyme mix (10µl 5X yPAP Reaction Buffer, 2µl of 600U/µl yPAP enzyme, 1µl 25mM ATP, 2µl 40U/µl Protector RNase Inhibitor, 35µl nuclease-free H2O) and incubating at 37ºC for 25 minutes without agitation. Nuclei were then washed with 500µl of nuclei suspension buffer (based on nuclei suspension buffer from Cao et al, Nature, 2019: 10mM Tris-HCl pH7.5, 10mM NaCl, 3mM MgCl2, 1% bovine serum albumin, 0.2U/µl Protector RNase Inhibitor, nuclease-free H2O) and pelleted by centrifugation at 1000 x g, at 4ºC, for 5 minutes. Nuclei were finally resuspended in 200µl nuclei suspension buffer, counted using a Countess 3 (Thermo Fisher Scientific) and the LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Fisher Scientific, #L3224), then diluted to the proper concentration. Nuclei for standard single-nucleus RNA-sequencing were processed similarly, but with no in situ polyadenylation step (counted immediately after wash buffer was added). 3,300 nuclei were then loaded onto the Chromium controller (10x Genomics) for a targeted capture of 2,000 nuclei. Libraries were generated using the standard Chromium v3 protocol. Final libraries were sequenced using the Illumina MiniSeq and Illumina NextSeq 500.