Drop-Seq

Bead Features

• Bead size uniformity:
  
  
• Barcode sequence complexity
• UMI sequence complexity
• Purity of V
• Uniformity of polyT

Custom Ability (Sequence, bead, and linker customization)

• Bead materials: Methacrylic polymer beads, Polystyrene beads (PS), PS beads cross linked with HVB
• Bead sizes: 5-micron, 10-micron, 20-micron, 20–40 -micron
• Sequence complexity: The various ratio of nucleotides in the cell barcode, UMI, and constant sequences can be specified
• Linkers attachment: Photo Cleavable, C6-Disulfide linkers, addition of dU bases (for enzymatic cleavage)
• Sequence Length: Up to 120-mer
• Beads to capture different sequences simultaneously: Beads can be designed to capture RNA/DNA using more than one handle. For example, mature transcripts using poly A tail and other species of RNA containing a shared stretch of sequences. RNA/DNA can also be captured using random 6-mer. Custom ratio of different capture sequence: Beads designed to capture RNA/DNA using more than one handle can contain desired ration of handles
• Free 3′ end vs free 5′ end: Capture sequence in the beads can contain free 3′ end (the cDNA resulting from Reverse Transcription is covalently attached to the beads) or free 5′ end (the cDNA resulting from Reverse Transcription is not covalently attached to the beads)
• Terminal and internal modifications: A wide array of nucleotide modifications are available for internal or terminal nucleotides, enabling various functionality

Specification of the Drop-Seq beads

For more information, please visit: http://mccarrolllab.com/dropseq/

Sequencing: Each Drop-Seq bead contains an extended UMI of 8N and V (9 total bases). As such, please adjust the Read 1 length of your sequencing accordingly.

Storage Instructions

Upon receipt, gently wash the beads twice with 30 mL of 100% EtOH (200 proof, for molecular biology). Invert several times to mix. Centrifuge the beads after each wash for 2 min at 500 g. (Caution: avoid vortexing the beads. Make sure no agitation is done which can cause breakage of beads.) Wash 2x with 30 mL of TE/TW (10 mM Tris pH 8.0, 1 mM EDTA, 0.01% Tween). Invert several times to mix. Centrifuge for 2 min at 500 g.

Resuspend the beads in 10 mL TE/TW and pass through a 10 µm filter (BD Falcon, cat #352360) into a 50 mL Falcon tube. It is preferable to suspend the beads in 50 mL TE/TW buffer for 10 µmol scale beads.

The beads should then be counted with a reliable hemocytometer. It is advisable to aliquot the beads into multiple tubes based on the number of experiments to prevent repeated cool and thaw cycles. Resuspended beads should be stored strictly between 2–4 degrees C.

Comprehensive Quality Control Test

References

1. Macosko, E., Basu, A., Satija, R. et al. Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell 161, 1202–1214 (2015). https://doi.org/10.1016/j.cell.2015.05.002
2. Rodriques, S., Stickels, R., Goeva, A. et al. Slide-seq: A scalable technology for measuring genome-wide expression at high spatial resolution. Science 363, 1463–1467 (2019). https://doi.org/10.1126/science.aaw1219
3. Gierahn, T., Wadsworth, M., Hughes, T. et al. Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput. Nat Methods 14 395–398 (2017). https://doi.org/10.1038/nmeth.4179

Resources

Brochure