JAX SCBL workflow for scRNA-seq of human placenta
William F Flynn, Paul Gabriel, Diane Luo, Jessica Grassmann, Elise Courtois, Paul Robson
Abstract
Described here is the workflow used by the JAX Single Cell Biology lab to generate single cell transcriptomic libraries from human placenta samples collected at the UCSD HuBMAP Female Reproductive Tissue Mapping Center.
Steps
Tissue collection
Tissue is collected and processed as soon as possible after Cesarean section or vaginal delivery according to the following protocol:
Human Placenta Tissue Collection and Preservation Methods - UCSD Female Reproductive TMC
Tissue is then stored in MACS storage buffer for overnight shipping.
Sample preparation
Upon receiving the sample, single cells are isolated through a digestion with cold active protease as described here:
Human endometrium and endometriosis tissue dissociation for single-cell RNA sequencing
The viability and cell concentration for each sample is assessed via a Countess FL II or LUNA FX7 automated cell counter.
The single cell suspension is then loaded onto one lane of the appropriate 10x Genomics gene expression chip as follows:
| A | B | C | D |
|---|---|---|---|
| Chip | Approx Cells Loaded | Approx. Cells Targeted | Compatiable Instrument |
| Chip G | 12,000 | 6,000 | Chromium Controller, Xi, X |
| Chip M | 40,000 | 20,000 | Chromium X |
Library Generation
The resulting loading, running, and library construction is performed according to the appropriate 10x Genomics protocol:
Sequencing
We have typically targeted 100,000 read pairs/cell for human placenta sample, resulting in target library sizes of roughly 600M-2,000M read pairs. These are multiplexed on a NovaSeq 6000 S4 lane using v1.5 reagents and the reverse complement workflow in a 28/8/0/90 or 28/8/8/90 configuration.
