SingleCell

From Genome Technology Core (GTC) wiki - Sequencing and Microarray
Jump to: navigation, search

We offer two types of single-cell sequencing: SMART-Seq2, and 10X Genomics.

10X Genomics

Scale – up to 10,000 cells per library

Input – fresh cultures < ~30 minutes old

Output – Counts - one read (close to the polyA tail) per RNA molecule

https://www.10xgenomics.com/single-cell/

Each 10X Genomics Single Cell 3’ RNA-seq library is derived from a pool of freshly-cultured, live cells, with up to 10,000 cells per sample. A single run can prepare up to 8 pools simultaneously, allowing for the preparation of up to 80,000 cells per processing run. The Chromium Controller is an instrument which combines individual cells and gel beads (GEMs) containing barcoded oligos into droplets prior to cDNA synthesis, so that each cell is represented by its own unique barcode prior to lysis and pooling. The barcoded DNA is then used to prepare a standard Illumina library. The end result is a single library containing data for each individual cell.

The user provides a freshly collected cell suspension on the day of processing. You must coordinate an appointment time with the Genome Core to allow for immediate processing after cell collection and counting. The user is responsible for obtaining cell counts and percent viability prior to arrival for the appointment. Detailed protocols are available from the Genome Core.

Please refer to our FAQ section for commonly asked questions: http://genomecore.wi.mit.edu/index.php/Faq#10X_Single_Cell_RNA

Please review the 10X FAQ on Single Cell Seq: https://kb.10xgenomics.com/hc/en-us/categories/201931746-General-Single-Cell-RNA-seq


SMART-Seq2

Scale – up to 96 cells per plate/library

Input – sorted cells in lysis buffer, frozen after lysis

Output – Counts - Multiple reads across full length of the molecule

SMART-Seq2 is based on a paper published in Nature Protocols (Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2, Nature Protocols, 9, no 1, (2014)). The protocol is derived from the Takara Bio “SMARTer” method, which uses a template switching oligo during RT to generate long, double stranded cDNA fragments from plates containing sorted cells, with one cell per well. This cDNA is then modified using Illumina Nextera XT technology, which uses a transposase to fragment the cDNA and ligate adapters for barcoding and amplification. The resulting single-cell libraries are typically pooled (96 to a pool) for Illumina sequencing, resulting in one final library sample per plate of cells.

The user should submit sorted cells in Eppendorf Twin-Tec plates, resuspended in a specific lysis buffer and frozen at -80ºC. Detailed instructions are available from the Genome Core. These plates are fairly stable, so the sorting does not need to be coordinated with the Genome Core prior to submission.